Methods, devices, and systems for analyte detection and analysis

ABSTRACT

Provided are systems and methods for analyte detection and analysis. A system can comprise an open substrate. The open substrate may be configured to rotate or otherwise move. The open substrate can comprise an array of individually addressable locations, with analytes immobilized thereto. The substrate may be spatially indexed to identify nucleic acid molecules from one or more sources, and/or sequences thereof, with the respective one or more sources. A solution comprising a plurality of probes may be directed across the array to couple at least one of the plurality of probes with at least one of the analytes to form a bound probe. A detector can be configured to detect a signal from the bound probe via scanning of the substrate while minimizing temperature fluctuations of the substrate or optical aberrations caused by bubbles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/445,798, filed Jun. 19, 2019 which claims the benefit ofU.S. Provisional Patent Application Ser. No. 62/818,549, filed Mar. 14,2019 and U.S. Provisional Patent Application Ser. No. 62/837,684, filedApr. 23, 2019.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/914,293, filed Oct. 11, 2019, U.S. ProvisionalPatent Application Ser. No. 62/837,684, filed Apr. 23, 2019 and U.S.Provisional Patent Application Ser. No. 62/818,549 filed Mar. 14, 2019,each of which applications is herein incorporated by reference in itsentirety for all purposes.

BACKGROUND

Biological sample processing has various applications in the fields ofmolecular biology and medicine (e.g., diagnosis). For example, nucleicacid sequencing may provide information that may be used to diagnose acertain condition in a subject and in some cases tailor a treatmentplan. Sequencing is widely used for molecular biology applications,including vector designs, gene therapy, vaccine design, industrialstrain design and verification. Biological sample processing may involvea fluidics system and/or a detection system.

SUMMARY

Despite the prevalence of biological sample processing systems andmethods, such systems and methods may have low efficiency that can betime-intensive and wasteful of valuable resources, such as reagents.Recognized herein is a need for methods and systems for sampleprocessing and/or analysis with high efficiency.

The present disclosure provides methods, devices, and systems for sampleprocessing and/or analysis. The methods, devices, and systems describedherein may comprise an open substrate, or use thereof. The opensubstrate may comprise one or more analytes thereon. For example, theone or more analytes may be coupled, attached, immobilized, or otherwiseassociated, directly or indirectly (e.g., via an intermediary object,such as a binder or linker) with the open substrate. The open substratemay comprise an array. In some instances, an environment of the opensubstrate, such as the local environment surrounding the open substrate,may be controlled, such as to facilitate one or more reactions, or oneor more detections. The methods, devices, and systems described hereinmay comprise immersion optics systems, or use thereof. An immersionoptics system may be configured to detect analytes, or activitiesthereof, on the open substrate. The methods, devices, and systemsdescribed herein may comprise spatial indexing of the open substrate, orarray thereof, or use thereof.

In various aspects, the present disclosure provides a method forscanning a surface, comprising: (a) using a scanner to scan a scanningfield comprising a portion of a surface, wherein the scanning field hasan orientation with respect to a rotational axis of the surface; and (b)rotating (i) the surface about the rotational axis of the surface and(ii) the scanning field about a rotational axis of the scanning field tosubstantially maintain the orientation of the scanning field withrespect to the rotational axis of the surface prior to, during, orsubsequent to translation of the surface and the scanning field relativeto one another.

In some embodiments, the orientation comprises a long axis of thescanning field, wherein the long axis is substantially parallel to orsubstantially coincident with a radial line passing through (i) therotational axis of the surface and (ii) the rotational axis of thescanning field. In some embodiments, in (b), the rotating substantiallymaintains the orientation prior to, during, or subsequent to translationof the surface or the scanning field. In some embodiments, thetranslation of the surface and the scanning field relative to oneanother comprises translating the scanning field or the surface along atranslation path, wherein a line comprising a net displacement along thetranslation path does not intersect both the scanning field and therotational axis of the surface. In some embodiments, the scanning fieldis rotated by rotating one or more elements of the scanner, which one ormore elements are selected from the group consisting of: an objective, alens, a prism, a mirror, a diffractive optical element, and a camera. Insome embodiments, the method further comprises rotating and translatingthe surface substantially simultaneously.

In some embodiments, the method further comprises illuminating a portionof the surface to provide an illumination field, wherein theillumination field at least partially overlaps the scanning field,wherein the illumination field is rotated such that the illuminationfield is oriented with respect to the rotational axis of the surface andis oriented with respect to the scanning field. In some embodiments, thescanning field and the illumination field are rotated together, andwherein a long axis of the illumination field is substantially parallelto a long axis of the scanning field. In some embodiments, theillumination field is rotated by rotating one or more elements of anillumination unit that generates the illumination field, which one ormore elements are selected from the group consisting of: an objective, alens, a prism, a mirror, a diffractive optical element, and a laser.

In some embodiments, the scanner comprises a camera, and wherein thescanning field is in optical communication with the camera comprising aline rate such that the camera takes an image when the scanning fieldhas advanced along the surface from a first location to a secondlocation, which second location is adjacent to the first location. Insome embodiments, the line rate is higher when the scanning field islocated farther from the rotational axis of the surface. In someembodiments, the camera is configured to take images at a frequency andthe surface is rotated relative to the objective at a variable angularvelocity, wherein the angular velocity is varied such that, at thefrequency, the camera takes an image when the scanning field is at afirst location and when the scanning field is at a second location,which second location is adjacent to the first location.

In some embodiments, the surface is rotated at a substantially constantangular velocity. In some embodiments, the surface is rotated such thata linear velocity of the surface and the scanning field relative to oneanother is substantially constant. In some embodiments, the scanningfield traces a nonlinear path on the surface.

In some embodiments, the scanner comprises a first objective and asecond objective in optical communication with the surface. In someembodiments, the first objective and the second objective are on a sameside of the surface with respect to a plane substantially normal to thesurface and containing the rotational axis of the surface. In someembodiments, the first objective and the second objective are onopposite sides of the surface with respect to a plane normal to thesurface and containing the rotational axis of the surface. In someembodiments, the first objective and the second objective tracealternating nonlinear paths. In some embodiments, the first objectivetraces a first path along the surface and the second objective traces asecond path along the surface, wherein the first path is closer to therotational axis than the second path. In some embodiments, the firstobjective traces a first path, the first path comprising a first widthcorresponding to a first width of the scanning field, and wherein thesecond objective traces a second path, the second path comprising asecond path width corresponding to a second width of a second scanningfield, wherein the first path width and the second path width overlap byno more than 30%. In some embodiments, wherein the first path firstobjective traces a first path, the first path comprising a first widthcorresponding to a first width of the scanning field, and wherein thesecond objective traces a second path, the second path comprising asecond path width corresponding to a second width of a second scanningfield, wherein the first path and the second path cover at least 50% ofthe surface.

In some embodiments, the scanner comprising the first objective and thesecond objective is translated relative to the surface along a lineextending radially from the rotational axis of the surface. In someembodiments, the first objective and second objective are translatedtogether toward or away from the rotational axis of the surface. In someembodiments, the first objective and second objective are translatedalong parallel paths of the surface.

In various aspects, the present disclosure provides a system,comprising: a scanner configured to scan a scanning field comprising aportion of a surface, wherein the scanning field has an orientation withrespect to a rotational axis of the surface; and a controller configuredto direct rotation of (i) the surface about the rotational axis of thesurface and (ii) the scanning field about a rotational axis of thescanning field to substantially maintain the orientation of the scanningfield with respect to the rotational axis of the surface prior to,during, or subsequent to translation of the surface and the scanningfield relative to one another.

In some embodiments, the scanning field is configured to rotate aboutthe rotational axis of the scanning field to adjust the orientation ofthe scanning field from a first orientation to a second orientationrelative to the rotational axis of the surface. In some embodiments, thesystem further comprises an illumination source configured to illuminatean illumination field comprising a second portion of the surface,wherein the illumination field and the scanning field at least partiallyoverlap one another. In some embodiments, an illumination profile of theillumination is configured to expand along a single axis. In someembodiments, the scanner comprises a first objective and a secondobjective configured to be in optical communication with the surface.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows a computer control system that is programmed or otherwiseconfigured to implement methods provided herein;

FIG. 2 shows a flowchart for an example of a method for sequencing anucleic acid molecule;

FIG. 3 shows a system for sequencing a nucleic acid molecule;

FIG. 4A shows a system for sequencing a nucleic acid molecule in a firstvertical level;

FIG. 4B shows a system for sequencing a nucleic acid molecule in asecond vertical level;

FIG. 5A shows a first example of a system for sequencing a nucleic acidmolecule using an array of fluid flow channels;

FIG. 5B shows a second example of a system for sequencing a nucleic acidmolecule using an array of fluid flow channels;

FIG. 6 shows a computerized system for sequencing a nucleic acidmolecule;

FIG. 7 illustrates a system with different environmental conditions inan open substrate system.

FIG. 8A-FIG. 8D illustrate schemes for line-scan cameras. FIG. 8Aillustrates rows of pixels for a time delay and integration (TDI)line-scan camera. FIG. 8B illustrates a trilinear pixel scheme for acolor line-scan camera including red (R), green (G), and blue (B)pixels. FIG. 8C and FIG. 8D illustrate bilinear pixel schemes for acolor line-scan camera including red, green, and blue pixels.

FIG. 9 shows an optical system for continuous area scanning of asubstrate during rotational motion of the substrate;

FIG. 10A shows an optical system for imaging a substrate duringrotational motion of the substrate using tailored optical distortions;

FIG. 10B shows an optical system for imaging a substrate duringrotational motion of the substrate using tailored optical distortions;

FIG. 10C shows an example of induced tailored optical distortions usinga cylindrical lens;

FIG. 11A illustrates schematically a scheme for expanding a laser beamto provide a laser line.

FIG. 11B illustrates schematically a scheme for expanding a laser beamto provide a laser line.

FIG. 11C shows an optical system for shaping a laser beam;

FIG. 12A-FIG. 12C illustrate schemes for detection of signals emitted bya material coupled to an open substrate. FIG. 12A illustrates a schemein which an open substrate rotates and a detector system remainsstationary during detection. FIG. 12B illustrates a scheme in which anopen substrate remains stationary and a detector system rotates duringdetection. FIG. 12C illustrates a scheme in which an open substraterotates during delivery and dispersal of a solution to the opensubstrate (left panel) and remains stationary during detection with arotating detector system (right panel).

FIG. 13A shows a first example of an interleaved spiral imaging scan;

FIG. 13B shows a second example of an interleaved imaging scan;

FIG. 13C shows an example of a nested imaging scan;

FIG. 14 shows a configuration for a nested circular imaging scan;

FIG. 15 shows a cross-sectional view of an immersion optical system;

FIG. 16 illustrates schematically an exemplary temperature gradientduring optical imaging.

FIG. 17A-FIG. 17B illustrate schematically exemplary methods to regulatetemperature of the substrate.

FIG. 17C-FIG. 17D illustrate schematically exemplary methods to regulatetemperature of the substrate.

FIG. 17E illustrates schematically exemplary methods to regulatetemperature of the substrate.

FIG. 18 illustrates schematically bubble formation in a fluid.

FIG. 19 illustrates schematically an adapter for an optical imagingsystem.

FIG. 20A illustrates schematically an exemplary method to displacebubbles, showing a substrate with a fluid dispensed thereto.

FIG. 20B illustrates schematically an exemplary method to displacebubbles, showing an optical imaging objective in contact with the fluid.

FIG. 21 illustrates schematically a method for dispensing and removingimmersion fluid onto a substrate.

FIG. 22A-FIG. 22B illustrate schematically a method for trapping bubblesand exemplary adapters for optical imaging objectives.

FIG. 23A shows an architecture for a system comprising a stationary axissubstrate and moving fluidics and optics;

FIG. 23B shows an architecture for a system comprising a translatingaxis substrate and stationary fluidics and optics;

FIG. 23C shows an architecture for a system comprising a plurality ofstationary substrates and moving fluidics and optics;

FIG. 23D shows an architecture for a system comprising a plurality ofmoving substrates on a rotary stage and stationary fluidics and optics;

FIG. 23E shows an architecture for a system comprising a plurality ofstationary substrates and moving optics;

FIG. 23F shows an architecture for a system comprising a plurality ofmoving substrates and stationary fluidics and optics;

FIG. 23G shows an architecture for a system comprising a plurality ofmoving substrates and stationary fluidics and optics;

FIG. 23H shows an architecture for a system comprising a plurality ofsubstrates moved between a plurality of processing bays;

FIG. 23I shows an architecture for a system comprising a plurality ofimaging heads scanning with shared translation and rotational axes andindependently rotating fields;

FIG. 23J shows an architecture for a system comprising a plurality ofimaging heads scanning with shared translation and rotational axes andindependently rotating fields;

FIG. 23K shows an architecture for a system comprising multiple spindlesscanning with a shared optical detection system;

FIG. 24 shows an architecture for a system comprising a plurality ofrotating spindles;

FIG. 25 shows a flowchart for an example of a method for processing ananalyte;

FIG. 26 shows a first example of a system for isolating an analyte; and

FIG. 27 shows a second example of a system for isolating an analyte.

FIG. 28 shows examples of control systems to compensate for velocitygradients during scanning.

FIG. 29A shows motion of a substrate relative to two imaging headslocated on the same side of an axis of rotation of the substrate.

FIG. 29B shows motion of a substrate relative to two imaging headslocated on opposite sides of an axis of rotation of the substrate.

FIG. 29C shows motion of a substrate relative to three imaging heads.

FIG. 29D shows motion of a substrate relative to four imaging heads.

FIG. 29E shows motion of a substrate relative to four imaging heads.

FIG. 29F shows motion of a substrate relative to four imaging heads.

FIG. 29G shows motion of a substrate relative to four imaging heads.

FIG. 30A shows successive ring paths of two imaging heads located on thesame side of an axis of rotation of a substrate.

FIG. 30B shows successive ring paths of two imaging heads located onopposite sides of an axis of rotation of a substrate.

FIG. 30C shows staggered ring paths of two imaging heads located on thesame side of an axis of rotation of a substrate.

FIG. 30D shows staggered ring paths of two imaging heads located onopposite sides of an axis of rotation of a substrate.

FIG. 31A shows rotating scan directions of imaging heads due tonon-radial motion of a substrate.

FIG. 31B shows rotating scan directions of imaging heads due tonon-radial motion of a substrate.

FIG. 32 shows a flowchart for an example of a method for analytedetection or analysis.

FIG. 33 illustrates schematically an axis of rotation and relativetranslation of a surface and an axis of rotation of an imaging field;

FIG. 34A illustrates schematically an optical system for rotating animaging field;

FIG. 34B illustrates schematically an optical system for rotating animaging field;

FIG. 34C illustrates schematically an optical system for rotating animaging field;

FIG. 35A shows an example of imaging head positioning for optimalscanning efficiency;

FIG. 35B shows an example of imaging head positioning for optimalscanning efficiency;

FIG. 35C shows an example of imaging head positioning for optimalscanning efficiency;

FIG. 36A-FIG. 36B illustrate schematically methods for processing abiological analyte.

FIG. 37A-FIG. 37G illustrate different examples of cross-sectionalsurface profiles of a substrate.

FIG. 38A-FIG. 38D illustrate a method of making anoligonucleotide-coated surface resistant to nucleic acid contaminants.

FIG. 39A-FIG. 39B illustrate two examples of spatial loading schemes.

FIG. 40 illustrates multiplex sample processing schemes.

FIG. 41 illustrates schematically an exemplary optical layout;

FIG. 42 shows an example of an image generated by imaging a substratewith an analyte immobilized thereto.

FIG. 43 shows an example of data obtained from a diagnostic procedure.

FIG. 44 shows example data of a diagnostic procedure. Panels A-F showspatial plots of diagnostic metrics computed on scanned images atdifferent individually addressable locations.

FIG. 45A shows example data of flow-based sequencing.

FIG. 45B-FIG. 45C illustrate exemplary data from processed images.

FIG. 46A shows a plot of aligned genomic reads. FIG. 46B shows alignedcoverage distribution over a reference genome.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “processing an analyte,” as used herein, generally refers toone or more stages of interaction with one more sample substances.Processing an analyte may comprise conducting a chemical reaction,biochemical reaction, enzymatic reaction, hybridization reaction,polymerization reaction, physical reaction, any other reaction, or acombination thereof with, in the presence of, or on, the analyte.Processing an analyte may comprise physical and/or chemical manipulationof the analyte. For example, processing an analyte may comprisedetection of a chemical change or physical change, addition of orsubtraction of material, atoms, or molecules, molecular confirmation,detection of the presence of a fluorescent label, detection of a Forsterresonance energy transfer (FRET) interaction, or inference of absence offluorescence. The term “analyte” may refer to molecules, cells,biological particles, or organisms. In some instances, a molecule may bea nucleic acid molecule, antibody, antigen, peptide, protein, or otherbiological molecule obtained from or derived from a biological sample.An analyte may originate from, and/or be derived from, a biologicalsample, such as from a cell or organism. An analyte may be synthetic.

The term “sequencing,” as used herein, generally refers to a process forgenerating or identifying a sequence of a biological molecule, such as anucleic molecule. Such sequence may be a nucleic acid sequence, whichmay include a sequence of nucleic acid bases. Sequencing may be singlemolecule sequencing or sequencing by synthesis, for example. Sequencingmay be performed using template nucleic acid molecules immobilized on asupport, such as a flow cell or one or more beads.

The term “biological sample,” as used herein, generally refers to anysample from a subject or specimen. The biological sample can be a fluidor tissue from the subject or specimen. The fluid can be blood (e.g.,whole blood), saliva, urine, or sweat. The tissue can be from an organ(e.g., liver, lung, or thyroid), or a mass of cellular material, suchas, for example, a tumor. The biological sample can be a feces sample,collection of cells (e.g., cheek swab), or hair sample. The biologicalsample can be a cell-free or cellular sample. Examples of biologicalsamples include nucleic acid molecules, amino acids, polypeptides,proteins, carbohydrates, fats, or viruses. In an example, a biologicalsample is a nucleic acid sample including one or more nucleic acidmolecules, such as deoxyribonucleic acid (DNA) and/or ribonucleic acid(RNA). The nucleic acid molecules may be cell-free or cell-free nucleicacid molecules, such as cell free DNA or cell free RNA. The nucleic acidmolecules may be derived from a variety of sources including human,mammal, non-human mammal, ape, monkey, chimpanzee, reptilian, amphibian,avian, or plant sources. Further, samples may be extracted from varietyof animal fluids containing cell free sequences, including but notlimited to blood, serum, plasma, vitreous, sputum, urine, tears,perspiration, saliva, semen, mucosal excretions, mucus, spinal fluid,amniotic fluid, lymph fluid and the like. Cell free polynucleotides maybe fetal in origin (via fluid taken from a pregnant subject) or may bederived from tissue of the subject itself.

The term “subject,” as used herein, generally refers to an individualfrom whom a biological sample is obtained. The subject may be a mammalor non-mammal. The subject may be an animal, such as a monkey, dog, cat,bird, or rodent. The subject may be a human. The subject may be apatient. The subject may be displaying a symptom of a disease. Thesubject may be asymptomatic. The subject may be undergoing treatment.The subject may not be undergoing treatment. The subject can have or besuspected of having a disease, such as cancer (e.g., breast cancer,colorectal cancer, brain cancer, leukemia, lung cancer, skin cancer,liver cancer, pancreatic cancer, lymphoma, esophageal cancer or cervicalcancer) or an infectious disease. The subject can have or be suspectedof having a genetic disorder such as achondroplasia, alpha-1 antitrypsindeficiency, antiphospholipid syndrome, autism, autosomal dominantpolycystic kidney disease, Charcot-Marie-tooth, cri du chat, Crohn'sdisease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome,Duchenne muscular dystrophy, factor V Leiden thrombophilia, familialhypercholesterolemia, familial Mediterranean fever, fragile x syndrome,Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly,Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonicdystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta,Parkinson's disease, phenylketonuria, Poland anomaly, porphyria,progeria, retinitis pigmentosa, severe combined immunodeficiency, sicklecell disease, spinal muscular atrophy, Tay-Sachs, thalassemia,trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGRsyndrome, or Wilson disease.

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidsequence,” “nucleic acid fragment,” “oligonucleotide” and“polynucleotide,” as used herein, generally refer to a polynucleotidethat may have various lengths, such as either deoxyribonucleotides ordeoxyribonucleic acids (DNA) or ribonucleotides or ribonucleic acids(RNA), or analogs thereof. Non-limiting examples of nucleic acidsinclude DNA, RNA, genomic DNA or synthetic DNA/RNA or coding ornon-coding regions of a gene or gene fragment, loci (locus) defined fromlinkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA,ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA),micro-RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branchednucleic acids, plasmids, vectors, isolated DNA of any sequence, andisolated RNA of any sequence. A nucleic acid molecule can have a lengthof at least about 10 nucleic acid bases (“bases”), 20 bases, 30 bases,40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40kb, 50 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 1 megabase (Mb), ormore. A nucleic acid molecule (e.g., polynucleotide) can comprise asequence of four natural nucleotide bases: adenine (A); cytosine (C);guanine (G); and thymine (T) (uracil (U) for thymine (T) when thepolynucleotide is RNA). A nucleic acid molecule may include one or morenonstandard nucleotide(s), nucleotide analog(s) and/or modifiednucleotide(s).

Nonstandard nucleotides, nucleotide analogs, and/or modified analogs mayinclude, but are not limited to, diaminopurine, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid(v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine, ethynyl nucleotide bases, 1-propynyl nucleotidebases, azido nucleotide bases, phosphoroselenoate nucleic acids and thelike. In some cases, nucleotides may include modifications in theirphosphate moieties, including modifications to a triphosphate moiety.Additional, non-limiting examples of modifications include phosphatechains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8,9, 10 or more phosphate moieties), modifications with thiol moieties(e.g., alpha-thio triphosphate and beta-thiotriphosphates) ormodifications with selenium moieties (e.g., phosphoroselenoate nucleicacids). Nucleic acid molecules may also be modified at the base moiety(e.g., at one or more atoms that typically are available to form ahydrogen bond with a complementary nucleotide and/or at one or moreatoms that are not typically capable of forming a hydrogen bond with acomplementary nucleotide), sugar moiety or phosphate backbone. Nucleicacid molecules may also contain amine-modified groups, such asaminoallyl-dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) toallow covalent attachment of amine reactive moieties, such asN-hydroxysuccinimide esters (NHS). Alternatives to standard DNA basepairs or RNA base pairs in the oligonucleotides of the presentdisclosure can provide higher density in bits per cubic mm, highersafety (resistant to accidental or purposeful synthesis of naturaltoxins), easier discrimination in photo-programmed polymerases, or lowersecondary structure. Nucleotide analogs may be capable of reacting orbonding with detectable moieties for nucleotide detection.

The term “nucleotide,” as used herein, generally refers to anynucleotide or nucleotide analog. The nucleotide may be naturallyoccurring or non-naturally occurring. The nucleotide analog may be amodified, synthesized or engineered nucleotide. The nucleotide analogmay not be naturally occurring or may include a non-canonical base. Thenaturally occurring nucleotide may include a canonical base. Thenucleotide analog may include a modified polyphosphate chain (e.g.,triphosphate coupled to a fluorophore). The nucleotide analog maycomprise a label. The nucleotide analog may be terminated (e.g.,reversibly terminated). The nucleotide analog may comprise analternative base.

The terms “amplifying,” “amplification,” and “nucleic acidamplification” are used interchangeably and generally refer togenerating one or more copies of a nucleic acid or a template. Forexample, “amplification” of DNA generally refers to generating one ormore copies of a DNA molecule. Moreover, amplification of a nucleic acidmay be linear, exponential, or a combination thereof. Amplification maybe emulsion based or may be non-emulsion based. Non-limiting examples ofnucleic acid amplification methods include reverse transcription, primerextension, polymerase chain reaction (PCR), ligase chain reaction (LCR),helicase-dependent amplification, asymmetric amplification, rollingcircle amplification, recombinase polymerase reaction (RPA), andmultiple displacement amplification (MDA). Where PCR is used, any formof PCR may be used, with non-limiting examples that include real-timePCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR,emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hotstart PCR, inverse PCR, methylation-specific PCR, miniprimer PCR,multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetricinterlaced PCR and touchdown PCR. Moreover, amplification can beconducted in a reaction mixture comprising various components (e.g., aprimer(s), template, nucleotides, a polymerase, buffer components,co-factors, etc.) that participate or facilitate amplification. In somecases, the reaction mixture comprises a buffer that permits contextindependent incorporation of nucleotides. Non-limiting examples includemagnesium-ion, manganese-ion and isocitrate buffers. Additional examplesof such buffers are described in Tabor, S. et al. C.C. PNAS, 1989, 86,4076-4080 and U.S. Pat. Nos. 5,409,811 and 5,674,716, each of which isherein incorporated by reference in its entirety.

The terms “dispense” and “disperse” may be used interchangeably herein.In some cases, dispensing may comprise dispersing and/or dispersing maycomprise dispensing. Dispensing generally refers to distributing,depositing, providing, or supplying a reagent, solution, or otherobject, etc. Dispensing may comprise dispersing, which may generallyrefer to spreading.

Useful methods for clonal amplification from single molecules includerolling circle amplification (RCA) (Lizardi et al., Nat. Genet.19:225-232 (1998), which is incorporated herein by reference), bridgePCR (Adams and Kron, Method for Performing Amplification of Nucleic Acidwith Two Primers Bound to a Single Solid Support, Mosaic Technologies,Inc. (Winter Hill, Mass.); Whitehead Institute for Biomedical Research,Cambridge, Mass., (1997); Adessi et al., Nucl. Acids Res. 28:E87 (2000);Pemov et al., Nucl. Acids Res. 33:e11(2005); or U.S. Pat. No. 5,641,658,each of which is incorporated herein by reference), polony generation(Mitra et al., Proc. Natl. Acad. Sci. USA 100:5926-5931 (2003); Mitra etal., Anal. Biochem. 320:55-65(2003), each of which is incorporatedherein by reference), and clonal amplification on beads using emulsions(Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), whichis incorporated herein by reference) or ligation to bead-based adapterlibraries (Brenner et al., Nat. Biotechnol. 18:630-634 (2000); Brenneret al., Proc. Natl. Acad. Sci. USA 97:1665-1670 (2000)); Reinartz, etal., Brief Funct. Genomic Proteomic 1:95-104 (2002), each of which isincorporated herein by reference).

The term “detector,” as used herein, generally refers to a device thatis capable of detecting a signal, including a signal indicative of thepresence or absence of one or more incorporated nucleotides orfluorescent labels. The detector may detect multiple signals. The signalor multiple signals may be detected in real-time during, substantiallyduring a biological reaction, such as a sequencing reaction (e.g.,sequencing during a primer extension reaction), or subsequent to abiological reaction. In some cases, a detector can include opticaland/or electronic components that can detect signals. The term“detector” may be used in detection methods. Non-limiting examples ofdetection methods include optical detection, spectroscopic detection,electrostatic detection, electrochemical detection, acoustic detection,magnetic detection, and the like. Optical detection methods include, butare not limited to, light absorption, ultraviolet-visible (UV-vis) lightabsorption, infrared light absorption, light scattering, Rayleighscattering, Raman scattering, surface-enhanced Raman scattering, Miescattering, fluorescence, luminescence, and phosphorescence.Spectroscopic detection methods include, but are not limited to, massspectrometry, nuclear magnetic resonance (NMR) spectroscopy, andinfrared spectroscopy. Electrostatic detection methods include, but arenot limited to, gel-based techniques, such as, for example, gelelectrophoresis. Electrochemical detection methods include, but are notlimited to, electrochemical detection of amplified product afterhigh-performance liquid chromatography separation of the amplifiedproducts.

The term “continuous area scanning,” as used herein, generally refers toarea scanning in rings, spirals, or arcs on a rotating substrate usingan optical imaging system and a detector. Continuous area scanning mayscan a substrate or array along a nonlinear path. Alternatively or inaddition, continuous area scanning may scan a substrate or array along alinear or substantially linear path. The detector may be a continuousarea scanning detector. The scanning direction may be substantially θ inan (R, θ) coordinate system in which the object rotation motion is in aθ direction. Across any field of view on the object (substrate) imagedby a scanning system, the apparent velocity may vary with the radialposition (R) of the field point on the object as

${R\frac{d\theta}{dt}}.$Continuous area scanning detectors may scan at the same rate for allimage positions and therefore may not be able to operate at the correctscan rate for all imaged points in a curved (or arcuate or non-linear)scan. Therefore, the scan may be corrupted by velocity blur for imagedfield points moving at a velocity different than the scan velocity.Continuous rotational area scanning may comprise an optical detectionsystem or method that makes algorithmic, optical, and/or electroniccorrections to substantially compensate for this tangential velocityblur, thereby reducing this scanning aberration. For example, thecompensation is accomplished algorithmically by using an imageprocessing algorithm that deconvolves differential velocity blur atvarious image positions corresponding to different radii on the rotatingsubstrate to compensate for differential velocity blur. In some cases,the camera or scanner may apply or use a blur to compensate fordifferential velocity blur.

In another example, the compensation is accomplished by using ananamorphic magnification gradient. This may serve to magnify thesubstrate in one axis (anamorphic magnification) by different amounts attwo or more substrate positions transverse to the scan direction. Theanamorphic magnification gradient may modify the imaged velocities ofthe two or more positions to be substantially equal thereby compensatingfor tangential velocity differences of the two positions on thesubstrate. This compensation may be adjustable to account for differentvelocity gradients across the field of view at different radii on thesubstrate.

The imaging field of view may be segmented into two or more regions,each of which can be electronically controlled to scan at a differentrate. These rates may be adjusted to the mean projected object velocitywithin each region. The regions may be optically defined using one ormore beam splitters or one or more mirrors. The two or more regions maybe directed to two or more detectors. The regions may be defined assegments of a single detector.

The term “continuous area scanning detector,” as used herein, generallyrefers to an imaging array sensor capable of continuous integration overa scanning area wherein the scanning is electronically synchronized tothe image of an object in relative motion. A continuous area scanningdetector may comprise a time delay and integration (TDI) charge coupleddevice (CCD), Hybrid TDI, or complementary metal oxide semiconductor(CMOS) pseudo TDI device. For example, a continuous area scanningdetector may comprise a TDI line-scan camera.

The term “open substrate”, as used herein, generally refers to asubstantially planar substrate in which a single active surface isphysically accessible at any point from a direction normal to thesubstrate. Substantially planar may refer to planarity at a micrometerlevel or nanometer level. Alternatively, substantially planar may referto planarity at less than a nanometer level or greater than a micrometerlevel (e.g., millimeter level).

The term “anamorphic magnification”, as used herein, generally refers todifferential magnification between two axes of an image. An anamorphicmagnification gradient may comprise differential anamorphicmagnification in a first axis across a displacement in the second axis.The magnification in the second axis may be unity or any other valuethat is substantially constant over the field.

The term “field of view”, as used herein, generally refers to the areaon the sample or substrate that is optically mapped to the active areaof the detector.

Processing an Analyte Using an Open Substrate

Prior microfluidic systems have utilized substrates containing numerouslong, narrow channels. The typical flow cell geometry for suchsubstrates introduces a need to compromise between two competingrequirements: 1) minimizing volume to minimize reagent usage; and 2)maximizing effective hydraulic diameter to minimize flow time. Thistrade-off may be especially important for washing operations, which mayrequire large wash volumes and thus long amounts of time to complete.The tradeoff is illustrated by the Poiseuille equation that dictatesflow in the laminar regime and is thus inherent to microfluidic systemsthat utilize such flow cell geometries. Such flow cell geometries mayalso be susceptible to contamination. Because such flow cell geometriesallow for a finite, limited number of channels in the microfluidicsystems, such finite number of channels may be shared between aplurality of different mixtures comprising different analytes, reagents,agents, and/or buffers. Contents of fluids flowing through the samechannels may be contaminated.

Described herein are devices, systems, and methods for processinganalytes using open substrates or flow cell geometries that can addressat least the abovementioned problems. The devices, systems and methodsmay be used to facilitate any application or process involving areaction or interaction between an analyte and a fluid (e.g., a fluidcomprising reagents, agents, buffers, other analytes, etc.). Suchreaction or interaction may be chemical (e.g., polymerase reaction) orphysical (e.g., displacement). The systems and methods described hereinmay benefit from higher efficiency, such as from faster reagent deliveryand lower volumes of reagents required per surface area. The systems andmethods described herein may avoid contamination problems common tomicrofluidic channel flow cells that are fed from multiport valves whichcan be a source of carryover from one reagent to the next. The devices,systems, and methods may benefit from shorter completion time, use offewer resources (e.g., various reagents), and/or reduced system costs.The open substrates or flow cell geometries may be used to process anyanalyte, such as but not limited to, nucleic acid molecules, proteinmolecules, antibodies, antigens, cells, and/or organisms, as describedherein. The open substrates or flow cell geometries may be used for anyapplication or process, such as, but not limited to, sequencing bysynthesis, sequencing by ligation, amplification, proteomics, singlecell processing, barcoding, and sample preparation, as described herein.

The systems and methods may utilize a substrate comprising an array(such as a planar array) of individually addressable locations. Eachlocation, or a subset of such locations, may have immobilized thereto ananalyte (e.g., a nucleic acid molecule, a protein molecule, acarbohydrate molecule, etc.). For example, an analyte may be immobilizedto an individually addressable location via a support, such as a bead. Aplurality of analytes immobilized to the substrate may be copies of atemplate analyte. For example, the plurality of analytes may havesequence homology. In other instances, the plurality of analytesimmobilized to the substrate may be different. The plurality of analytesmay be of the same type of analyte (e.g., a nucleic acid molecule) ormay be a combination of different types of analytes (e.g., nucleic acidmolecules, protein molecules, etc.). One or more surfaces of thesubstrate may be exposed to a surrounding open environment, andaccessible from such surrounding open environment. For example, thearray may be exposed and accessible from such surrounding openenvironment. In some cases, as described elsewhere herein, thesurrounding open environment may be controlled and/or confined in alarger controlled environment.

Reagents may be dispensed to the substrate to multiple locations, and/ormultiple reagents may be dispensed to the substrate to a singlelocation, via different mechanisms. In some cases, dispensing (tomultiple locations and/or of multiple reagents to a single location) maybe achieved via relative motion of the substrate and the dispenser(e.g., nozzle). For example, a reagent may be dispensed to the substrateat a first location, and thereafter travel to a second locationdifferent from the first location due to forces (e.g., centrifugalforces, centripetal forces, inertial forces, etc.) caused by motion ofthe substrate. In another example, a reagent may be dispensed to areference location, and the substrate may be moved relative to thereference location such that the reagent is dispensed to multiplelocations of the substrate. In some cases, dispensing (to multiplelocations and/or of multiple reagents to a single location) may beachieved without relative motion between the substrate and thedispenser. For example, multiple dispensers may be used to dispensereagents to different locations, and/or multiple reagents to a singlelocation, or a combination thereof (e.g., multiple reagents to multiplelocations). In another example, an external force (e.g., involving apressure differential), such as wind, may be applied to one or moresurfaces of the substrate to direct reagents to different locationsacross the substrate. In another example, the method for dispensingreagents (e.g., to multiple locations and/or of multiple reagents to asingle location) may comprise vibration. In such an example, reagentsmay be distributed or dispensed onto a single region or multiple regionsof the substrate (or a surface of the substrate). The substrate (or asurface thereof) may then be subjected to vibration, which may spreadthe reagent to different locations across the substrate (or thesurface). Alternatively or in conjunction, the method may comprise usingmechanical, electric, physical, or other means to dispense reagents tothe substrate. For example, the solution may be dispensed onto asubstrate and a physical scraper (e.g., a squeegee) may be used tospread the dispensed material or spread the reagents to differentlocations and/or to obtain a desired thickness or uniformity across thesubstrate. Beneficially, such flexible dispensing may be achievedwithout contamination of the reagents. In some instances, where a volumeof reagent is dispensed to the substrate at a first location, andthereafter travels to a second location different from the firstlocation, the volume of reagent may travel in a path or paths, such thatthe travel path or paths are coated with the reagent. In some cases,such travel path or paths may encompass a desired surface area (e.g.,entire surface area, partial surface area(s), etc.) of the substrate.

Reagents may be dispensed over the uncovered surface or substrate at adesired flow rate. The flow rate of fluid dispensing may be about (e.g.,at ambient temperature, or about 25 degrees Celsius) 1 picoliter/min, 10picoliters/min, 100 picoliters/min, 1 nanoliter/min, 10 nanoliters/min,100 nanoliters/min, 1 microliter/min, 10 microliters/min, 100microliters/min, 1 milliliter/min, 10 milliliters/min, 100milliliters/min, up to 1 liter/min. The flow rate of fluid dispensingmay be between any of these flow rates. The flow rate of fluiddispensing may be at least any of these flow rates. Alternatively, theflow rate of fluid dispensing may be at most any of these flow rates.The flow rate may be tuned according to desired properties of thereagent or solution layer (e.g., thickness).

Solutions may comprise reagents, samples, or any useful substance. Thesolution may comprise a fluid that has desirable flow properties. Forexample, the fluid may have a temperature-variable viscosity. Thesolution may comprise a non-Newtonian fluid. The solution may comprise apower law fluid, such as a shear-thinning (thixotropic) orshear-thickening fluid. The solution may comprise a Newtonian fluid.

In some cases, the substrate may be rotatable about an axis. Theanalytes may be immobilized to the substrate during rotation. Reagents(e.g., nucleotides, antibodies, washing reagents, enzymes, etc.) may bedispensed onto the substrate prior to or during rotation (for instance,spun at a high rotational velocity) of the substrate to coat the arraywith the reagents and allow the analytes to interact with the reagents.For example, when the analytes are nucleic acid molecules and when thereagents comprise nucleotides, the nucleic acid molecules mayincorporate or otherwise react with (e.g., transiently bind) one or morenucleotides. In another example, when the analytes are protein moleculesand when the reagents comprise antibodies, the protein molecules maybind to or otherwise react with one or more antibodies. In anotherexample, when the reagents comprise washing reagents, the substrate(and/or analytes on the substrate) may be washed of any unreacted(and/or unbound) reagents, agents, buffers, and/or other particles.

In some cases, the substrate may be movable in any vector or direction,as described elsewhere herein. For example, such motion may benon-linear (e.g., in rotation about an axis). In another example, suchmotion may be linear. In other examples, the motion may be a hybrid oflinear and non-linear motion. The analytes may be immobilized to thesubstrate during any such motion. Reagents (e.g., nucleotides,antibodies, washing reagents, enzymes, etc.) may be dispensed onto thesubstrate prior to or during motion of the substrate to facilitatecoating of the array with the reagents and allow the analytes tointeract with the reagents.

In some cases, where the substrate is rotatable, high speed coatingacross the substrate may be achieved via tangential inertia directingunconstrained spinning reagents in a partially radial direction (thatis, away from the axis of rotation) during rotation, a phenomenoncommonly referred to as centrifugal force. High speed rotation mayinvolve a rotational speed of at least 1 revolution per minute (rpm), atleast 2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm, at least50 rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm, at least1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, at least 10,000 rpm,or greater. This mode of directing reagents across a substrate may beherein referred to as centrifugal or inertial pumping. Inertial forcesmay direct unconstrained reagents across the substrate in any directionduring any type of motion (e.g., rotational motion, non-rotationalmotion, linear motion, non-linear motion, accelerated motion, etc.) ofthe substrate.

One or more signals (such as optical signals) may be detected from adetection area on the substrate prior to, during, or subsequent to, thedispensing of reagents to generate an output. For example, the outputmay be an intermediate or final result obtained from processing of theanalyte. Signals may be detected in multiple instances. The dispensing,rotating (or other motion), and/or detecting operations, in any order(independently or simultaneously), may be repeated any number of timesto process an analyte. In some instances, the substrate may be washed(e.g., via dispensing washing reagents) between consecutive dispensingof the reagents. One or more detection operations can be performedwithin a desired time frame. For example, the detection operation can beperformed within about 1 minute, 50 seconds, 40 seconds, 30 seconds, 20seconds, 10 seconds or less than 10 seconds. In some instances, at leasttwo detection operations can be performed within 1 minute, 50 seconds,40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 secondsetc. In some instances, at least three detection operations can beperformed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20seconds, 10 seconds or less than 10 seconds.

Provided herein is a method for processing a biological analyte,comprising providing a substrate comprising an array having immobilizedthereto the biological analyte, wherein the substrate is rotatable withrespect to a central axis. In some instances, the array can be a planararray. In some instances, the array can be an array of wells. In someinstances, the substrate can be textured and/or patterned. The methodcan comprise directing a solution across the substrate and bringing thesolution in contact with the biological analyte during rotation of thesubstrate. The solution may be directed in a radial direction (e.g.,outwards) with respect to the substrate to coat the substrate andcontact the biological analytes immobilized to the array. In someinstances, the solution may comprise a plurality of probes. In someinstances, the solution may be a washing solution. The method cancomprise subjecting the biological analyte to conditions sufficient toconduct a reaction between at least one probe of the plurality of probesand the biological analyte. The reaction may generate one or moresignals from the at least one probe coupled to the biological analyte.The method can comprise detecting one or more signals, thereby analyzingthe biological analyte.

In other cases, provided herein is a method for processing a biologicalanalyte, comprising providing a substrate comprising an array havingimmobilized thereto the biological analyte, wherein the substrate ismovable with respect to a reference axis. The method can comprisedirecting a solution across the substrate and bringing the solution incontact with the biological analyte during motion of the substrate. Insome instances, the motion can be linear. In some instances, the motioncan be non-linear. In some instances, the motion can be a hybrid betweenlinear and non-linear motion.

In other cases, provided herein is a method for processing a biologicalanalyte, comprising providing a substrate comprising an array havingimmobilized thereto the biological analyte. In some instances, themethod can comprise dispensing a solution to two different locations onthe substrate and/or array. In some instances, the method can comprisedispensing multiple solutions to a single location on the substrateand/or array, such as using multiple dispensers. In some instances, themethod can comprise dispensing multiple solutions to multiple locationson the substrate and/or array. In some instances, the method cancomprise dispensing a single solution to a single location. Thesubstrate may be in relative motion with respect to one or moredispensers. The substrate may be stationary with respect to one or moredispensers. One or more dispensing operations can be performed within adesired time frame. For example, the dispensing operation can beperformed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20seconds, 10 seconds or less than 10 seconds. In some instances, at leasttwo dispensing operations can be performed within 1 minute, 50 seconds,40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 secondsetc. In some instances, at least three dispensing operations can beperformed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20seconds, 10 seconds or less than 10 seconds.

Any operation or process of one or more methods disclosed herein may beperformed within a desired time frame. In some instances, a combinationof two or more operations or processes disclosed herein may be performedwithin a desired time frame. For example, the dispensing operation andthe detection method may both be performed within 1 minute, 50 seconds,40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds.In some instances, at least two dispensing and detection operations canbe performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20seconds, 10 seconds or less than 10 seconds etc. In some instances, atleast three dispensing and detection operations can be performed within1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds orless than 10 seconds.

One or more methods disclosed herein may obviate the need for barcodingof analytes (e.g., nucleic acid molecules), which may be time-consumingand expensive. For example, alternative or in addition to barcoding, thesubstrate and/or array may be spatially indexed to identify theanalytes, as described elsewhere herein. One or more methods disclosedherein may obviate the need for unique barcoding of individual analytes(e.g., individual nucleic acid molecules).

The biological analyte may be any analyte that comes from a sample. Forinstance, the biological analyte may be a macromolecule, e.g., a nucleicacid molecule, a carbohydrate, a protein, a lipid, etc. The biologicalanalyte may comprise multiple macromolecular groups, e.g.,glycoproteins, proteoglycans, ribozymes, liposomes, etc. The biologicalanalyte may be an antibody, antibody fragment, or engineered variantthereof, an antigen, a cell, a peptide, a polypeptide, etc. In somecases, the biological analyte comprises a nucleic acid molecule. Thenucleic acid molecule may comprise at least about 10, 100, 1000, 10,000,100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000 or morenucleotides. Alternatively or in addition, the nucleic acid molecule maycomprise at most about 1,000,000,000, 100,000,000, 10,000,000,1,000,000, 100,000, 10,000, 1000, 100, 10 or fewer nucleotides. Thenucleic acid molecule may have a number of nucleotides that is within arange defined by any two of the preceding values. In some cases, thenucleic acid molecule may also comprise a common sequence, to which anN-mer may bind. An N-mer may comprise 1, 2, 3, 4, 5, or 6 nucleotidesand may bind the common sequence. In some cases, the nucleic acidmolecules may be amplified to produce a colony of nucleic acid moleculesattached to the substrate or attached to beads that may associate withor be immobilized to the substrate. In some instances, the nucleic acidmolecules may be attached to beads and subjected to a nucleic acidreaction, e.g., amplification, to produce a clonal population of nucleicacid molecules attached to the beads.

Nucleic acid molecules in any given nucleic acid sample may eachcomprise a key sequence. The key sequence may be a synthetic sequence.In some instances, the key sequence may be at most about 6 bases inlength, 5 bases in length, 4 bases in length, 3 bases in length, 2 basesin length, or 1 base in length. Alternatively, the key sequence may begreater than 6 bases in length. The key sequence may be indicative ofthe originating sample. For example, the key sequence may be unique to asample such that each sample of a plurality of samples has a unique keysequence. Individual analytes in a single sample may share the same keysequence. Alternatively, each sample may have a unique key sequencebetween its immediate neighboring samples when loaded onto thesubstrate. Beneficially, where two samples comprising different keysequences are loaded into adjacent or otherwise proximate regions on thesubstrate, nucleic acid molecules originating from different samples maybe readily differentiated based on the different key sequences evenwhere there is cross-contamination between regions (e.g., outlyingnucleic acid molecules that are inadvertently loaded onto a neighboringregion due to spillover, etc.) with relatively short reads (e.g., whichare much shorter than reads of unique barcode sequences that areconfigured to differentiate individual molecules).

The substrate may be a solid substrate. The substrate may entirely orpartially comprise one or more of rubber, glass, silicon, a metal suchas aluminum, copper, titanium, chromium, or steel, a ceramic such astitanium oxide or silicon nitride, a plastic such as polyethylene (PE),low-density polyethylene (LDPE), high-density polyethylene (HDPE),polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS),polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrilebutadiene styrene (ABS), polyacetylene, polyamides, polycarbonates,polyesters, polyurethanes, polyepoxide, polymethyl methacrylate (PMMA),polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamineformaldehyde (MF), urea-formaldehyde (UF), polyetheretherketone (PEEK),polyetherimide (PEI), polyimides, polylactic acid (PLA), furans,silicones, polysulfones, any mixture of any of the preceding materials,or any other appropriate material. The substrate may be entirely orpartially coated with one or more layers of a metal such as aluminum,copper, silver, or gold, an oxide such as a silicon oxide (Si_(x)O_(y),where x, y may take on any possible values), a photoresist such as SU8,a surface coating such as an aminosilane or hydrogel, polyacrylic acid,polyacrylamide dextran, polyethylene glycol (PEG), or any combination ofany of the preceding materials, or any other appropriate coating. Theone or more layers may have a thickness of at least 1 nanometer (nm), atleast 2 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 50nm, at least 100 nm, at least 200 nm, at least 500 nm, at least 1micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500μm, or at least 1 millimeter (mm). The one or more layers may have athickness that is within a range defined by any two of the precedingvalues. A surface of the substrate may be modified to comprise any ofthe binders or linkers described herein. A surface of the substrate maybe modified to comprise active chemical groups, such as amines, esters,hydroxyls, epoxides, and the like, or a combination thereof. In someinstances, such binders, linkers, active chemical groups, and the likemay be added as an additional layer or coating to the substrate.

The substrate may have the general form of a cylinder, a cylindricalshell or disk, a rectangular prism, or any other geometric form. Thesubstrate may have a thickness (e.g., a minimum dimension) of at least100 μm, at least 200 μm, at least 500 μm, at least 1 mm, at least 2 mm,at least 5 mm, or at least 10 mm. The substrate may have a thicknessthat is within a range defined by any two of the preceding values. Thesubstrate may have a first lateral dimension (such as a width for asubstrate having the general form of a rectangular prism or a radius fora substrate having the general form of a cylinder) of at least 1 mm, atleast 2 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 50mm, at least 100 mm, at least 200 mm, at least 500 mm, or at least 1,000mm. The substrate may have a first lateral dimension that is within arange defined by any two of the preceding values. The substrate may havea second lateral dimension (such as a length for a substrate having thegeneral form of a rectangular prism) or at least 1 mm, at least 2 mm, atleast 5 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100mm, at least 200 mm, at least 500 mm, or at least 1,000 mm. Thesubstrate may have a second lateral dimension that is within a rangedefined by any two of the preceding values.

A surface of the substrate may be planar. A surface of the substrate maybe uncovered and may be exposed to an atmosphere. Alternatively or inaddition, a surface of the substrate may be textured or patterned. Forexample, the substrate may comprise grooves, troughs, hills, and/orpillars. The substrate may define one or more cavities (e.g.,micro-scale cavities or nano-scale cavities). The substrate may defineone or more channels. The substrate may have a regular textures and/orpatterns across the surface of the substrate. For example, the substratemay have regular geometric structures (e.g., wedges, cuboids, cylinders,spheroids, hemispheres, etc.) above or below a reference level of thesurface. Alternatively, the substrate may have irregular textures and/orpatterns across the surface of the substrate. For example, the substratemay have any arbitrary structure above or below a reference level of thesubstrate. In some instances, a texture of the substrate may comprisestructures having a maximum dimension of at most about 100%, 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% of the total thickness of thesubstrate or a layer of the substrate. In some instances, the texturesand/or patterns of the substrate may define at least part of anindividually addressable location on the substrate. A textured and/orpatterned substrate may be substantially planar.

For example, FIG. 37A-FIG. 37G illustrate different examples ofcross-sectional surface profiles of a substrate. FIG. 37A illustrates across-sectional surface profile of a substrate having a completelyplanar surface. FIG. 37B illustrates a cross-sectional surface profileof a substrate having semi-spherical troughs or grooves. FIG. 37Cillustrates a cross-sectional surface profile of a substrate havingpillars, or alternatively or in conjunction, wells. FIG. 37D illustratesa cross-sectional surface profile of a substrate having a coating. FIG.37E illustrates a cross-sectional surface profile of a substrate havingspherical particles. FIG. 37F illustrates a cross-sectional surfaceprofile of FIG. 37B, with a first type of binders seeded or associatedwith the respective grooves. FIG. 37G illustrates a cross-sectionalsurface profile of FIG. 37B, with a second type of binders seeded orassociated with the respective grooves.

The substrate may comprise an array. For instance, the array may belocated on a lateral surface of the substrate. The array may be a planararray. The array may have the general shape of a circle, annulus,rectangle, or any other shape. The array may comprise linear and/ornon-linear rows. The array may be evenly spaced or distributed. Thearray may be arbitrarily spaced or distributed. The array may haveregular spacing. The array may have irregular spacing. The array may bea textured array. The array may be a patterned array. The array maycomprise a plurality of individually addressable locations. Theindividually addressable locations may be arranged in any convenientpattern. For example, the individually addressable locations may berandomly oriented on the array. The plurality of individuallyaddressable locations may form separate radial regions around adisk-shaped substrate. The plurality of individually addressablelocations may form a square, rectangle, disc, circular, annulus,pentagonal, hexagonal, heptagonal, octagonal, array, or any otherpattern. One or more types of individually addressable locations may begenerated. The one or more types of individually addressable locationsmay form alternating regions of the different types of individuallyaddressable locations. The one or more types of individually addressablelocations may form blocked regions of the different types ofindividually addressable locations. For example, in cases when two types(A and B) of individually addressable locations are desired, theindividually addressable locations may be arrayed as alternating ABABAB,blocked AAABBB, or random, e.g. ABBAAB, AABBBA, BABBAA, etc. The typesof individually addressable locations may be arrayed in any usefulpattern, such as a square, rectangle, disc, annulus, pentagon, hexagon,radial pattern, etc. In some cases, the two types of individuallyaddressable locations may have different chemical, physical, and/orbiological properties (e.g., hydrophobicity, charge, color, topography,size, dimensions, geometry, etc.). For example, a first type ofindividually addressable location may bind a first type of biologicalanalyte but not a second type of biological analyte, and a second typeof individually addressable location may bind the second type ofbiological analyte but not the first type of biological analyte.

The analyte to be processed may be immobilized to the array. The arraymay comprise one or more binders described herein, such as one or morephysical or chemical linkers or adaptors, that are coupled to abiological analyte. For instance, the array may comprise a linker oradaptor that is coupled to a nucleic acid molecule. Alternatively or inaddition, the biological analyte may be coupled to a bead, which beadmay be immobilized to the array. In some cases, a subset of the arraymay not be coupled to a sample or analyte. For example, in substratesthat are configured to rotate about a central axis, the samples may notbe coupled to a plurality of individually addressable locations of thearray located near the central axis. In some cases, the array may becoupled to a sample or an analyte, but not all of the array may beprocessed. For example, the substrate may be coupled to a sample oranalyte (e.g., comprising nucleic acid molecules), but the region of thearray that is in proximity to the border of the array may not besubjected to further processing (e.g., detection).

The individually addressable locations may comprise locations ofanalytes or groups of analytes that are accessible for manipulation. Themanipulation may comprise placement, extraction, reagent dispensing,seeding, heating, cooling, or agitation. The extraction may compriseextracting individual analytes or groups of analytes. For instance, theextraction may comprise extracting at least 2, at least 5, at least 10,at least 20, at least 50, at least 100, at least 200, at least 500, orat least 1,000 analytes or groups of analytes. Alternatively or inaddition, the extraction may comprise extracting at most 1,000, at most500, at most 200, at most 100, at most 50, at most 20, at most 10, atmost 5, or at most 2 analytes or groups of analytes. The manipulationmay be accomplished through, for example, localized microfluidic, pipet,optical, laser, acoustic, magnetic, and/or electromagnetic interactionswith the analyte or its surroundings.

In some cases, the individually addressable locations may be indexed,e.g., spatially, such that the analyte immobilized or coupled to eachindividually addressable location may be identified. In someembodiments, the individually addressable locations are indexed bydemarcating part of the substrate. In some embodiments, the surface ofthe substrate is demarcated using etching. In some embodiments, thesurface of the substrate is demarcated using a notch in the surface. Insome embodiments, the surface of the substrate is demarcated using a dyeor ink. In some embodiments, the surface of the substrate is demarcatedby depositing a topographical mark on the surface. In some embodiments,a sample, such as a control nucleic acid sample, may be used todemarcate the surface of the substrate. As will be appreciated, acombination of positive demarcations and negative demarcations (lackthereof) may be used to index the individually addressable locations. Insome instances, a single reference point or axis (e.g., singledemarcation) may be used to index all individually addressablelocations. In some embodiments, each of the individually addressablelocations is indexed. In some embodiments, a subset of the individuallyaddressable locations is indexed. In some embodiments, the individuallyaddressable locations are not indexed, and a different region of thesubstrate is indexed.

Individually addressable locations, or individual regions comprising theindividually addressable locations, may be indexed, or otherwisedistinguished. In some instances, the individually addressablelocations, or individual regions may be distinguished solely by sampleloading (e.g., without physical demarcations). In some instances, asingle region may be distinguished from other regions. In someinstances, a single type of region may be distinguished from other typesof regions. For example, different types of regions may comprisedifferent types of analytes or different sets of samples. For example, afirst type of region (“A”) may comprise a first set of samples (or firsttype of sample), and a second type of region (“B”) may comprise a secondset of samples (or second type of sample). The substrate may comprise aset of multiple region A's and a set of multiple region B's, wherein themultiple region A's are distinguishable from the multiple region B's.Different samples may be loaded onto the different types of regions in apredetermined spatial configuration to allow such distinction.

In some cases, a key or barcode sequence on the sample may be used todistinguish and/or index the spatial locations, originating sample, or acombination thereof. For example, nucleic acid molecules in any givennucleic acid sample may each comprise a key sequence. The key sequencemay be a synthetic sequence. The key sequence may be at most about 6bases in length, 5 bases in length, 4 bases in length, 3 bases inlength, 2 bases in length, or 1 base in length. Alternatively, the keysequence may be greater than 6 bases in length. The key sequence may beindicative of the originating sample. For example, the key sequence maybe unique to a sample such that each sample of a plurality of sampleshas a unique key sequence. Individual analytes of a single sample mayshare a common key sequence. Alternatively, each sample may have aunique key sequence between its immediate neighboring samples whenloaded onto the substrate. Beneficially, where two samples comprisingdifferent key sequences are loaded into adjacent or otherwise proximateregions on the substrate, nucleic acid molecules originating fromdifferent samples may be readily differentiated based on the differentkey sequences even where there is cross-contamination between regions(e.g., outlying nucleic acid molecules that are inadvertently loadedonto a neighboring region due to spillover, etc.) with relatively shortreads (e.g., which are much shorter than reads of barcode sequences thatare configured to differentiate individual molecules).

In some cases, spatial separation of analytes may be used to augment orreplace the use of key or barcode sequences. For example, FIG. 40illustrates schemes for analysis of analytes in a single region or inmultiple regions, including 7, 15, and 96 regions. For example, as shownin FIG. 40, analytes may be distributed across the entire surface (upperleft, “1-plex”) distributed in discrete regions, including 7 regions(upper right, “7-plex”), 15 regions (lower left, “15-plex”), or 96regions (lower right, “96-plex”). Analytes may be distributed in about5, about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 45, about 50, about 60, about 70, about 80, about 90, about 100,about 200, about 300, about 400, or about 500 regions. Analytes may bedistributed in from 5 to 10, from 10 to 15, from 15 to 20, from 20 to25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, from 45to 50, from 50 to 60, from 60 to 70, from 70 to 80, from 80 to 90, from90 to 100, from 100 to 200, from 200 to 300, from 300 to 400, or from400 to 500 regions. In some cases, each region may contain a differentanalyte.

In some cases, different types of regions may be used for sampleprocessing. A first type of region (“A”) may comprise a first set ofsamples (or first type of sample), and a second type of region (“B”) maycomprise a second set of samples (or second type of sample). The firsttype of region and the second type of region may be disposed apart fromone another in an ordered fashion, as described elsewhere herein. Insome cases, the first type of region and the second type of region maybe disposed at a distance from a reference axis of the substrate. Forexample, the first type of region may be disposed at least 1 micrometer,10 micrometers, 100 micrometers, 1 millimeter, 10 millimeters, 100millimeters, 1 centimeter, 10 centimeters, 100 centimeters or more fromthe reference axis of the substrate. Similarly, the second type ofregion may be disposed at a distance from a reference axis of thesubstrate. For example, the first type of region may be disposed atleast 1 micrometer, 10 micrometers, 100 micrometers, 1 millimeter, 10millimeters, 100 millimeters, 1 centimeter, 10 centimeters, 100centimeters or more from the reference axis of the substrate. Both typesof regions may be disposed at least 1 micrometer, 10 micrometers, 100micrometers, 1 millimeter, 10 millimeters, 100 millimeters, 1centimeter, 10 centimeters, 100 centimeters or more from the referenceaxis of the substrate.

For example, FIG. 39A-FIG. 39B illustrate two examples of spatialloading schemes. In FIG. 39A, a substrate comprises two types of regions“A”s and “B”s which are disposed in radially alternating fashion withrespect to a central axis of the substrate. In FIG. 39B, a substratecomprises two types of regions “A”s and “B”s which are disposed intriangularly alternating fashion across the substrate. Sample locationsmay be determined by loading a first set of samples to the A regions,wherein the first set of samples comprises a plurality of beads coupledto analytes of the first set of samples, and detecting the plurality ofbeads and/or analytes and their locations on the substrate, and thenloading the second set of samples to the B regions, wherein the secondset of samples comprises a plurality of beads coupled to analytes of thesecond set of samples, and detecting the plurality of beads and/oranalytes and their locations on the substrate. Each sample in the firstset of samples and the second set of samples may be associated with alabel (e.g., fluorescent dye). Even though the first set of samples isprimarily loaded onto the A regions, there may be some crossovers inwhich stray beads from the first set of samples are immobilized to the Bregions. Even though the second set of samples is primarily loaded ontothe B regions, there may be some crossovers in which stray beads fromthe second set of samples are immobilized to the A regions. Thelocations of the analytes of the first set of samples, including thecross-over beads, can be determined from the first image. The locationsof the analytes of the second set of samples, including the cross-overbeads, can be determined from the second image. Beneficially, where thesame type of fluorescent dye identifies analytes of two differentsamples (“P” and “Q”), and “P” is deposited to an A region, and “Q” isdeposited to a B region, based on the type of region where thefluorescent signal is detected, one may identify if the analyte is ofthe “P” sample or the “Q” sample. The different regions may bealternating. The plurality of regions may form any pattern, such as atriangular, square, rectangle, disc, circular, annulus, pentagonal,hexagonal, heptagonal, octagonal, array, or any other pattern. Theplurality of regions may form irregular patterns. The plurality ofregions may be discrete regions that are not patterned. The plurality ofregions may be interleaved, interspersed, non-contiguous, and/ordifferent in size.

While examples herein describe two types of regions, there may be anynumber of regions (e.g., alternating regions) to achieve the alternatingspatial distinction described herein. For example, there may be at 1 atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10 regions.

While examples herein generally describe the loading of two samples ortwo sets of samples, any number of samples, or sets of samples, may beimmobilized to the substrate. For example, the substrate may haveimmobilized thereto at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10samples, or sets of samples. In some cases, at least about 10, 100,1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000or more samples, or sets of samples, may be immobilized. Alternativelyor in addition, the substrate may comprise at most about 1,000,000,000,100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 1000, 100, 10 orfewer samples, or sets of samples. When the sample is a nucleic acidsample, at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10 nucleic acidsamples may be immobilized to the substrate. In some cases, at leastabout 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000,100,000,000, 1,000,000,000 or more nucleic acid samples may beimmobilized. Alternatively or in addition, the substrate may comprise atmost about 1,000,000,000, 100,000,000, 10,000,000, 1,000,000, 100,000,10,000, 1000, 100, 10 or fewer nucleic acid samples. Beneficially,multiple samples may be simultaneously processed on the same substrate,without needing to otherwise barcode the multiple samples (e.g., with acommon barcode sequence per sample) to distinguish them.

Indexing may be performed using a detection method and may be performedat any convenient or useful step. A substrate that is indexed, e.g.,demarcated, may be subjected to detection, such as optical imaging, tolocate the indexed locations, individually addressable locations, and/orthe biological analyte. Imaging may be performed using a detection unit.Imaging may be performed using one or more sensors. Imaging may not beperformed using the naked eye. The substrate that is indexed may beimaged prior to loading of the biological analyte. Following loading ofthe biological analyte onto the individually addressable locations, thesubstrate may be imaged again, e.g. to determine occupancy or todetermine the positioning of the biological analyte relative to thesubstrate. In some cases, the substrate may be imaged after iterativecycles of nucleotide addition (or other probe or other reagent), asdescribed elsewhere herein. The indexing of the substrate and knowninitial position (individually addressable location) of the biologicalanalyte may allow for analysis and identification of the sequenceinformation for each individually addressable location and/or position.Additionally, spatial indexing may allow for identification of errorsthat may occur, e.g., sample contamination, sample loss, etc.

In some cases, indexing may be performed to identify, process, and/oranalyze more than one type of biological analyte, as described above.For example, a first type of biological analyte, which may be labeled,may be loaded onto a first set of locations within a substrate. Thesubstrate may be imaged for a first indexing step of the first type ofbiological analyte. A second type of biological analyte may be loadedonto a second set of locations within the substrate and imaged for asecond indexing step of the second type of biological analyte. In somecases, the second type of biological analyte may be labeled in a waysuch that the second type of biological analyte is distinguishable fromthe first type of biological analyte. Alternatively, the first type ofbiological analyte and the second type of biological analyte may belabeled in substantially the same detectable manner (e.g., same dye),and the first and second images may be processed to generate adifferential image, wherein overlapping signals are attributed to thelocations of the first type of biological analyte and different signalsare attributed to the locations of the second type of biologicalanalyte. Alternatively, the first type of biological analyte and thesecond type of biological analyte may be labeled by cleavable (orotherwise removable) labels or tags (e.g., fluorescent tags), and thelabel cleaved after each imaging operation, such that only the relevantanalyte locations are imaged at each imaging operation. Henceforth, thesubstrate may be analyzed and all of the locations comprising the firstbiological analyte may be attributed to the first biological analyte,and all of the locations comprising the second biological analyte may beattributed to the second analyte. In some cases, labeling of the firstand second analyte may not be necessary, and the attribution of thelocation to either the first or second analyte may be performed based onspatial location alone. This process may be repeated for any number ortypes of biological analytes.

The array may be coated with binders. For instance, the array may berandomly coated with binders. Alternatively, the array may be coatedwith binders arranged in a regular pattern (e.g., in linear arrays,radial arrays, hexagonal arrays etc.). The array may be coated withbinders on at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% of the number ofindividually addressable locations, or of the surface area of thesubstrate. The array may be coated with binders on a fraction ofindividually addressable locations, or of the surface areas of thesubstrate, that is within a range defined by any two of the precedingvalues. The binders may be integral to the array. The binders may beadded to the array. For instance, the binders may be added to the arrayas one or more coating layers on the array.

The binders may immobilize biological analytes through non-specificinteractions, such as one or more of hydrophilic interactions,hydrophobic interactions, electrostatic interactions, physicalinteractions (for instance, adhesion to pillars or settling withinwells), and the like. The binders may immobilize biological analytesthrough specific interactions. For instance, where the biologicalanalyte is a nucleic acid molecule, the binders may compriseoligonucleotide adaptors configured to bind to the nucleic acidmolecule. Alternatively or in addition, such as to bind other types ofanalytes, the binders may comprise one or more of antibodies,oligonucleotides, nucleic acid molecules, aptamers, affinity bindingproteins, lipids, carbohydrates, and the like. The binders mayimmobilize biological analytes through any possible combination ofinteractions. For instance, the binders may immobilize nucleic acidmolecules through a combination of physical and chemical interactions,through a combination of protein and nucleic acid interactions, etc. Thearray may comprise at least about 10, 100, 1000, 10,000, 100,000,1,000,000, 10,000,000, 100,000,000 or more binders. Alternatively or inaddition, the array may comprise at most about 100,000,000, 10,000,000,1,000,000, 100,000, 10,000, 1000, 100, 10 or fewer binders. The arraymay have a number of binders that is within a range defined by any twoof the preceding values. In some instances, a single binder may bind asingle biological analyte (e.g., nucleic acid molecule). In someinstances, a single binder may bind a plurality of biological analytes(e.g., plurality of nucleic acid molecules). In some instances, aplurality of binders may bind a single biological analyte. Thoughexamples herein describe interactions of binders with nucleic acidmolecules, the binders may immobilize other molecules (such asproteins), other particles, cells, viruses, other organisms, or thelike.

In some instances, each location, or a subset of such locations, mayhave immobilized thereto an analyte (e.g., a nucleic acid molecule, aprotein molecule, a carbohydrate molecule, etc.). In other instances, afraction of the plurality of individually addressable location may haveimmobilized thereto an analyte. A plurality of analytes immobilized tothe substrate may be copies of a template analyte. For example, theplurality of analytes (e.g., nucleic acid molecules) may have sequencehomology. In other instances, the plurality of analytes immobilized tothe substrate may not be copies. The plurality of analytes may be of thesame type of analyte (e.g., a nucleic acid molecule) or may be acombination of different types of analytes (e.g., nucleic acidmolecules, protein molecules, etc.).

In some instances, the array may comprise a plurality of types ofbinders. For example, the array may comprise different types of bindersto bind different types of analytes. For example, the array may comprisea first type of binders (e.g., oligonucleotides) configured to bind afirst type of analyte (e.g., nucleic acid molecules), and a second typeof binders (e.g., antibodies) configured to bind a second type ofanalyte (e.g., proteins), and the like. In another example, the arraymay comprise a first type of binders (e.g., first type ofoligonucleotide molecules) to bind a first type of nucleic acidmolecules and a second type of binders (e.g., second type ofoligonucleotide molecules) to bind a second type of nucleic acidmolecules, and the like. For example, the substrate may be configured tobind different types of analytes in certain fractions or specificlocations on the substrate by having the different types of binders inthe certain fractions or specific locations on the substrate.

A biological analyte may be immobilized to the array at a givenindividually addressable location of the plurality of individuallyaddressable locations. An array may have any number of individuallyaddressable locations. For instance, the array may have at least 1, atleast 2, at least 5, at least 10, at least 20, at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, at least 10,000, at least 20,000, at least 50,000, at least100,000, at least 200,000, at least 500,000, at least 1,000,000, atleast 2,000,000, at least 5,000,000, at least 10,000,000, at least20,000,000, at least 50,000,000, at least 100,000,000, at least200,000,000, at least 500,000,000, at least 1,000,000,000, at least2,000,000,000, at least 5,000,000,000, at least 10,000,000,000, at least20,000,000,000, at least 50,000,000,000, or at least 100,000,000,000individually addressable locations. The array may have a number ofindividually addressable locations that is within a range defined by anytwo of the preceding values. Each individually addressable location maybe digitally and/or physically accessible individually (from theplurality of individually addressable locations). For example, eachindividually addressable location may be located, identified, and/oraccessed electronically or digitally for mapping, sensing, associatingwith a device (e.g., detector, processor, dispenser, etc.), or otherwiseprocessing. As described elsewhere herein, each individually addressablelocation may be indexed. Alternatively, the substrate may be indexedsuch that each individually addressable location may be identifiedduring at least one step of the process. Alternatively or in addition,each individually addressable location may be located, identified,and/or accessed physically, such as for physical manipulation orextraction of an analyte, reagent, particle, or other component locatedat an individually addressable location.

Multiple biological analytes may be immobilized to the array atspatially discrete locations. Spatial separation of biological analytesmay be obtained using masks or barriers, as described elsewhere herein.Alternatively or in conjunction, biological analytes may be separatedusing different fluid compositions. In some cases, the fluidcompositions may be immiscible. For example, a first solution (e.g., anoil, organic solution, or other hydrophobic or oleophilic solution) maycomprise a first biological analyte, and a second solution (e.g., ahydrophilic, aqueous, polar or ionic solution) may comprise a secondbiological analyte. The first and second solutions may be immiscible.The substrate may be exposed to the first solution in defined regions,e.g., using a mask (e.g., covering or shielding the other regions of thesubstrate). In some cases, the first biological analyte associates withdefined regions (e.g., individually addressable locations), and thefirst solution may be removed from the substrate. The substrate may thenbe exposed to the second solution. The second biological analyte maythen associate with the unoccupied sites of the substrate.Alternatively, the substrate may be pre-treated such that biologicalanalytes may be loaded in discrete locations. In one non-limitingexample, the substrate may be patterned with discrete hydrophobic andhydrophilic regions (e.g., using photolithography, soft lithography,etching, etc.) that can attract or repel a subset of the biologicalanalytes. In another non-limiting example, an inert polymer such aspolyethylene glycol (PEG) may be patterned in discrete regions toprevent attachment or the biological analyte to the substrate in thediscrete regions.

Each individually addressable location may have the general shape orform of a circle, pit, bump, rectangle, or any other shape or form. Eachindividually addressable location may have a first lateral dimension(such as a radius for individually addressable locations having thegeneral shape of a circle or a width for individually addressablelocations having the general shape of a rectangle). The first lateraldimension may be at least 1 nanometer (nm), at least 2 nm, at least 5nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, atleast 200 nm, at least 500 nm, at least 1,000 nm, at least 2,000 nm, atleast 5,000 nm, or at least 10,000 nm. The first lateral dimension maybe within a range defined by any two of the preceding values. Eachindividually addressable location may have a second lateral dimension(such as a length for individually addressable locations having thegeneral shape of a rectangle). The second lateral dimension may be atleast 1 nanometer (nm), at least 2 nm, at least 5 nm, at least 10 nm, atleast 20 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least500 nm, at least 1,000 nm, at least 2,000 nm, at least 5,000 nm, or atleast 10,000 nm. The second lateral dimension may be within a rangedefined by any two of the preceding values. In some instances, eachindividually addressable locations may have or be coupled to a binder,as described herein, to immobilize an analyte thereto. In someinstances, only a fraction of the individually addressable locations mayhave or be coupled to a binder. In some instances, an individuallyaddressable location may have or be coupled to a plurality of binders toimmobilize an analyte thereto.

The individually addressable locations may be generated using a varietyof methods. In one embodiment, the method may comprise generation ofindividually addressable locations using one or more barriers. In someembodiments, the barrier may be removed during any convenient operation.For example, the barrier may be removed prior to or after coupling theanalyte to the individually addressable locations. The barrier may beremoved prior to or after loading of the solution comprising a pluralityof probes. The barrier may be removed prior to or after subjecting theanalyte to conditions sufficient to conduct a reaction between the probeand the analyte. The barrier may be removed prior to or after detectionof one or more signals from the coupled probe and analyte. The barriermay be removed prior to or after detection of the coupled probe andanalyte. The barrier may be removed prior to or after repeating any ofthe abovementioned processes. In some cases, the barriers may not beremoved.

The barrier may comprise a physical, chemical, biological, or any othertype of obstruction. In some embodiments, the barrier comprises aphysical obstruction. In one such example, a mold may be used, wherein aportion of the mold may obstruct the movement of fluid to a specifiedregion. The mold may be generated using a variety of means, such asinjection molding, machining, heat treatment, fiber spinning, joiningand bonding, casting, rolling, forging, 3D printing, etc. In someembodiments, the barrier may be configured to dissolve at any convenientstep. The barrier may be configured to dissolve, evaporate, or sublime.In some cases, the barrier may be melted and removed. In some cases,removal of the barrier or part of the barrier may be achieved using anair knife. In some cases, the barrier comprises a chemical obstruction.In some cases, the barrier comprises a polymer. The barrier may comprisepolyethylene glycol (PEG). In some cases, the barrier may comprise asolution. The solution may be viscous. The solution may have atemperature-variable viscosity. The solution may be a non-Newtonianfluid. The solution may be a power law fluid, such as a shear-thinning(e.g., thixotropic) or shear-thickening fluid. The solution may be aNewtonian fluid. In some embodiments, the barrier comprises a fluid thatis immiscible with a loading solution. In some cases, the barrier is ahydrophobic region on the substrate.

A mask may be additionally or alternatively used to prevent coupling ofthe sample and/or biological analyte with a region of the substrate.Alternatively or in conjunction, a subset of the individuallyaddressable locations comprising the biological analyte may be masked,e.g., to prevent coupling of the probe to the biological analyte. A maskmay comprise a barrier, such as a physical, chemical or biologicalbarrier. A mask may comprise a film with removed sections. In somecases, the mask may be interfaced with the substrate prior tointroduction of the biological analyte. In such cases, introduction ofthe biological analyte may allow for coupling of the biological analyteto exposed regions of the mask-substrate interface, whereas thenon-exposed regions may remain free of the biological analyte. At anyconvenient process, the substrate may be un-masked. Any combinations ofmasks may be used. For example, a first mask may be used to load a firstbiological analyte to a desired region. Subsequently, the first mask maybe removed, and a second mask may be used to load a second biologicalanalyte to a desired region. The first and second region may haveoverlapping regions or may remain spatially distinct. A barrier and maskmay be used in conjunction or separately.

The analytes bound to the individually addressable locations mayinclude, but are not limited to, molecules, cells, organisms, nucleicacid molecules, nucleic acid colonies, beads, clusters, polonies, DNAnanoballs, or any combination thereof (e.g., bead having attachedthereto one or more nucleic acid molecules). The bound analytes may beimmobilized to the array in a regular, patterned, periodic, random, orpseudo-random configuration, or any other spatial arrangement. In someembodiments, the analytes are bound to bead(s) which may then associatewith or be immobilized to the substrate or regions of the substrate(e.g., individually addressable locations). In some embodiments, theanalytes comprise a bead or a plurality of beads. In some cases, thebead or plurality of beads may comprise another analyte (e.g., nucleicacid molecule) or a clonal population of other analytes (e.g., a nucleicacid molecule that has been amplified on the bead). Such other analytesmay be attached or otherwise coupled to the bead. For example, ananalyte may comprise a plurality of beads, each bead having a clonalpopulation of nucleic acid molecules attached thereto. In some cases,the bead is magnetic, and application of a magnetic field or using amagnet may be used to direct the analytes or beads comprising theanalytes to the individually addressable locations. In some cases, afluid may be used to direct the analyte to the individually addressablelocations. The fluid may be a ferrofluid, and a magnet may be used todirect the fluid to the individually addressable locations. Theindividually addressable locations may alternatively or in conjunctioncomprise a material that is sensitive to a stimulus, e.g., thermal,chemical, or electrical or magnetic stimulus. For example, theindividually addressable location may comprise a photo-sensitive polymeror reagent that is activated when exposed to electromagnetic radiation.In some cases, a caged molecule may be used to reveal binding (e.g.,biotin) moieties on the substrate. Subsequent exposure to a particularwavelength of light may result in un-caging of the binding moieties. Abead, e.g. with streptavidin, comprising the analyte may then associatewith the uncaged binding moieties. In some cases, a subset of theindividually addressable locations may not contain beads. In such cases,blank beads may be added to the substrate. The blank beads may thenoccupy the regions that are unoccupied by an analyte. In some cases, theblank beads have a higher binding affinity or avidity for theindividually addressable locations than the beads comprising theanalyte. In some cases, unoccupied locations may be destroyed. In somecases, unoccupied locations may be subjected to a process to remove anyunbound analyte, e.g., aspiration, washing, air blasting etc. In somecases, the sample comprising the biological analyte may be loaded ontothe substrate using a device, e.g., a microfluidic device, closed flowcell, etc. The loaded biological analyte may then associate with or beimmobilized to the substrate or the individually addressable locationsof the substrate. In such cases, the device may be removed followingloading of the sample.

A biological analyte may be bound to any number of beads. Differentbiological analytes may be bound to any number of beads. The beads maybe unique (i.e., distinct from each other). Any number of unique beadsmay be used. For instance, at least about 10, 100, 1000, 10,000,100,000, 1,000,000, 10,000,000, 100,000,000 or more different beads maybe used. Alternatively or in addition, at most about 100,000,000,10,000,000, 1,000,000, 100,000, 10,000, 1000, 100, 10 or fewer differentbeads may be used. A number of different beads can be within a rangedefined by any two of the preceding values. The beads may bedistinguishable from one another using a property of the beads, such ascolor, reflectance, anisotropy, brightness, fluorescence, etc.

A sample may be diluted such that the approximate occupancy of theindividually addressable locations is controlled. A sample may bediluted at least to a dilution of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100,1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:10000,1:100000, 1:1000000, 1:10000000, 1:100000000. Alternatively, a samplemay be diluted at most to a dilution of A sample may be diluted at leastto a dilution of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300,1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:10000, 1:100000,1:1000000, 1:10000000, 1:100000000. A dilution between any of thesedilution values may also be used.

The substrate may be configured to rotate with respect to an axis. Insome instances, the systems, devices, and apparatus described herein mayfurther comprise a rotational unit configured to rotate the substrate.The rotational unit may comprise a motor and/or a rotor to rotate thesubstrate. Such motor and/or rotor may be mechanically connected to thesubstrate directly or indirectly via intermediary components (e.g.,gears, stages, actuators, discs, pulleys, etc.). The rotational unit maybe automated. Alternatively or in addition, the rotational unit mayreceive manual input. The axis of rotation may be an axis through thecenter of the substrate (e.g., as shown in FIG. 33). The axis may be anoff-center axis. For instance, the substrate may be affixed to a chuck(such as a vacuum chuck) of a spin coating apparatus. The substrate maybe configured to rotate with a rotational velocity of at least 1revolution per minute (rpm), at least 2 rpm, at least 5 rpm, at least 10rpm, at least 20 rpm, at least 50 rpm, at least 100 rpm, at least 200rpm, at least 500 rpm, at least 1,000 rpm, at least 2,000 rpm, at least5,000 rpm, or at least 10,000 rpm. The substrate may be configured torotate with a rotational velocity that is within a range defined by anytwo of the preceding values. The substrate may be configured to rotatewith different rotational velocities during different operationsdescribed herein. The substrate may be configured to rotate with arotational velocity that varies according to a time-dependent function,such as a ramp, sinusoid, pulse, or other function or combination offunctions. The time-varying function may be periodic or aperiodic.

The substrate may be configured to move in any vector with respect to areference point. In some instances, the systems, devices, and apparatusdescribed herein may further comprise a motion unit configured to movethe substrate. The motion unit may comprise any mechanical component,such as a motor, rotor, actuator, linear stage, drum, roller, pulleys,etc., to move the substrate. Such components may be mechanicallyconnected to the substrate directly or indirectly via intermediarycomponents (e.g., gears, stages, actuators, discs, pulleys, etc.). Themotion unit may be automated. Alternatively or in addition, the motionunit may receive manual input. The substrate may be configured to movewith any velocity. In some instances, the substrate may be configured tomove with different velocities during different operations describedherein. The substrate may be configured to move with a velocity thatvaries according to a time-dependent function, such as a ramp, sinusoid,pulse, or other function or combination of functions. The time-varyingfunction may be periodic or aperiodic.

A solution may be provided to the substrate prior to or during rotation(or other motion) of the substrate to centrifugally (or otherwiseinertially) direct the solution across the array. In some instances, thesolution may be provided to the planar array during rotation of thesubstrate in pulses, thereby creating an annular wave of the solutionmoving radially outward. In some instances, the solution may be providedto the planar array during other motion of the substrate in pulses,thereby creating a wave of the solution moving in a certain direction.The pulses may have periodic or non-periodic (e.g., arbitrary)intervals. A series of pulses may comprise a series of waves producing asurface-reagent exchange. The surface-reagent exchange may comprisewashing in which each subsequent pulse comprises a reduced concentrationof the surface reagent. The solution may have a temperature differentthan that of the substrate, thereby providing a source or sink ofthermal energy to the substrate or to an analyte located on thesubstrate. The thermal energy may provide a temperature change to thesubstrate or to the analyte. The temperature change may be transient.The temperature change may enable, disable, enhance, or inhibit achemical reaction, such as a chemical reaction to be carried out uponthe analyte. For example, the chemical reaction may comprisedenaturation, hybridization, or annealing of nucleic acid molecules. Thechemical reaction may comprise a step in a polymerase chain reaction(PCR), bridge amplification, or other nucleic acid amplificationreaction. The temperature change may modulate, increase, or decrease asignal detected from the analyte.

The array may be in fluid communication with at least one sample inlet(of a fluid channel). The array may be in fluid communication with thesample inlet via a non-solid gap, e.g., an air gap. In some cases, thearray may additionally be in fluid communication with at least onesample outlet. The array may be in fluid communication with the sampleoutlet via an airgap. The sample inlet may be configured to direct asolution to the array. The sample outlet may be configured to receive asolution from the array. The solution may be directed to the array usingone or more dispensing nozzles. For example, the solution may bedirected to the array using at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or at least 20dispensing nozzles. The solution may be directed to the array using anumber of nozzles that is within a range defined by any two of thepreceding values. In some cases, different reagents (e.g., nucleotidesolutions of different types, different probes, washing solutions, etc.)may be dispensed via different nozzles, such as to preventcontamination. Each nozzle may be connected to a dedicated fluidic lineor fluidic valve, which may further prevent contamination. A type ofreagent may be dispensed via one or more nozzles. The one or morenozzles may be directed at or in proximity to a center of the substrate.Alternatively, the one or more nozzles may be directed at or inproximity to a location on the substrate other than the center of thesubstrate. Alternatively or in combination, one or more nozzles may bedirected closer to the center of the substrate than one or more of theother nozzles. For instance, one or more nozzles used for dispensingwashing reagents may be directed closer to the center of the substratethan one or more nozzles used for dispensing active reagents. The one ormore nozzles may be arranged at different radii from the center of thesubstrate. Two or more nozzles may be operated in combination to deliverfluids to the substrate more efficiently. One or more nozzles may beconfigured to deliver fluids to the substrate as a jet, spray (or otherdispersed fluid), and/or droplets. One or more nozzles may be operatedto nebulize fluids prior to delivery to the substrate. For example, thefluids may be delivered as aerosol particles.

The solution may be dispensed on the substrate while the substrate isstationary; the substrate may then be subjected to rotation (or othermotion) following the dispensing of the solution. Alternatively, thesubstrate may be subjected to rotation (or other motion) prior to thedispensing of the solution; the solution may then be dispensed on thesubstrate while the substrate is rotating (or otherwise moving).

Rotation of the substrate may yield a centrifugal force (or inertialforce directed away from the axis) on the solution, causing the solutionto flow radially outward over the array. In this manner, rotation of thesubstrate may direct the solution across the array. Continued rotationof the substrate over a period of time may dispense a fluid film of anearly constant thickness across the array. The rotational velocity ofthe substrate may be selected to attain a desired thickness of a film ofthe solution on the substrate. The film thickness may be related to therotational velocity by equation (1):

$\begin{matrix}{{h(t)} = \frac{\sqrt{3\;{\mu/2}}}{\sqrt{{2\; t\;\rho\;\omega^{2}} - {3\mu\; C}}}} & (1)\end{matrix}$Here, h(t) is the thickness of the fluid film at time t, μ is theviscosity of the fluid, ω is the rotational velocity, and C is aconstant.

Alternatively or in combination, the viscosity of the solution may bechosen to attain a desired thickness of a film of the solution on thesubstrate. For instance, the rotational velocity of the substrate or theviscosity of the solution may be chosen to attain a film thickness of atleast 10 nanometers (nm), at least 20 nm, at least 50 nm, at least 100nm, at least 200 nm, at least 500 nm, at least 1 micrometer (μm), atleast 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50μm, at least 100 μm. at least 200 μm, at least 500 μm, or at least 1 mm.The rotational velocity of the substrate and/or the viscosity of thesolution may be chosen to attain a film thickness that is within a rangedefined by any two of the preceding values. The viscosity of thesolution may be controlled by controlling a temperature of the solution.The thickness of the film may be measured or monitored. Measurements ormonitoring of the thickness of the film may be incorporated into afeedback system to better control the film thickness. The thickness ofthe film may be measured or monitored by a variety of techniques. Forinstances, the thickness of the film may be measured or monitored bythin film spectroscopy with a thin film spectrometer, such as a fiberspectrometer.

In some instances, one or more factors such as the rotational velocityof the substrate, the acceleration of the substrate (e.g., the rate ofchange of velocity), viscosity of the solution, angle of dispensing(e.g., contact angle of a stream of reagents) of the solution, radialcoordinates of dispensing of the solution (e.g., on center, off center,etc.), temperature of the substrate, temperature of the solution, andother factors may be adjusted and/or otherwise optimized to attain adesired wetting on the substrate and/or a film thickness on thesubstrate, such as to facilitate uniform coating of the substrate. Suchoptimization may prevent the solution from exiting the substrate along arelatively focused stream or travel path such that the fluid onlycontacts the substrate at partial surface areas (as opposed to theentire surface area)—in such cases, a significantly larger volume ofreagents may have to be dispensed to achieve uniform and full coating ofthe substrate. Such optimization may also prevent the solution fromscattering or otherwise reflecting or bouncing off the substrate uponcontact and disturbing the surface fluid. Alternatively or inconjunction, the thickness of the solution may be adjusted usingmechanical, electric, physical, or other mechanisms. For example, thesolution may be dispensed onto a substrate and subsequently leveledusing, e.g., a physical scraper such as a squeegee, to obtain a desiredthickness of uniformity across the substrate.

The substrate or a surface thereof may comprise other features that aidin solution or reagent retention on the substrate or thicknessuniformity of the solution or reagent on the substrate. In some cases,the surface may comprise a raised edge (e.g., a rim) which may be usedto retain solution on the surface. The surface may comprise a rim nearthe outer edge of the surface, thereby reducing the amount of thesolution that flows over the outer edge.

The solution may be a reaction mixture comprising a variety ofcomponents. For example, the solution may comprise a plurality of probesconfigured to interact with the analyte. For example, the probes mayhave binding specificity to the analyte. In another example, the probesmay not have binding specificity to the analyte. A probe may beconfigured to permanently couple to the analyte. A probe may beconfigured to transiently couple to the analyte. For example, anucleotide probe may be permanently incorporated into a growing strandhybridized to a nucleic acid molecule analyte. Alternatively, anucleotide probe may transiently bind to the nucleic acid moleculeanalyte. Transiently coupled probes may be subsequently removed from theanalyte. Subsequent removal of the transiently coupled probes from ananalyte may or may not leave a residue (e.g., chemical residue) on theanalyte. A type of probe in the solution may depend on the type ofanalyte. A probe may comprise a functional group or moiety configured toperform specific functions. For example, a probe may comprise a label(e.g., dye). A probe may be configured to generate a detectable signal(e.g., optical signal), such as via the label, upon coupling orotherwise interacting with the analyte. In some instances, a probe maybe configured to generate a detectable signal upon activation (e.g.,application of a stimulus). In another example, a nucleotide probe maycomprise reversible terminators (e.g., blocking groups) configured toterminate polymerase reactions (until unblocked). The solution maycomprise other components to aid, accelerate, or decelerate a reactionbetween the probe and the analyte (e.g., enzymes, catalysts, buffers,saline solutions, chelating agents, reducing agents, other agents,etc.). In some instances, the solution may be a washing solution. Insome instances, a washing solution may be directed to the substrate tobring the washing solution in contact with the array after a reaction orinteraction between reagents (e.g., a probe) in a reaction mixturesolution with an analyte immobilized on the array. The washing solutionmay wash away any free reagents from the previous reaction mixturesolution.

A detectable signal, such as an optical signal (e.g., fluorescentsignal), may be generated upon reaction between a probe in the solutionand the analyte. For example, the signal may originate from the probeand/or the analyte. The detectable signal may be indicative of areaction or interaction between the probe and the analyte. Thedetectable signal may be a non-optical signal. For example, thedetectable signal may be an electronic signal. The detectable signal maybe detected by one or more sensors. For example, an optical signal maybe detected via one or more optical detectors in an optical detectionscheme described elsewhere herein. The signal may be detected duringrotation of the substrate. The signal may be detected followingtermination of the rotation. The signal may be detected while theanalyte is in fluid contact with the solution. The signal may bedetected following washing of the solution. In some instances, after thedetection, the signal may be muted, such as by cleaving a label from theprobe and/or the analyte, and/or modifying the probe and/or the analyte.Such cleaving and/or modification may be affected by one or morestimuli, such as exposure to a chemical, an enzyme, light (e.g.,ultraviolet light), or temperature change (e.g., heat). In someinstances, the signal may otherwise become undetectable by deactivatingor changing the mode (e.g., detection wavelength) of the one or moresensors, or terminating or reversing an excitation of the signal. Insome instances, detection of a signal may comprise capturing an image orgenerating a digital output (e.g., between different images).

The operations of directing a solution to the substrate and detection ofone or more signals indicative of a reaction between a probe in thesolution and an analyte in the array may be repeated one or more times.Such operations may be repeated in an iterative manner. For example, thesame analyte immobilized to a given location in the array may interactwith multiple solutions in the multiple repetition cycles. For eachiteration, the additional signals detected may provide incremental, orfinal, data about the analyte during the processing. For example, wherethe analyte is a nucleic acid molecule and the processing is sequencing,additional signals detected for each iteration may be indicative of abase in the nucleic acid sequence of the nucleic acid molecule. Theoperations may be repeated at least 1, at least 2, at least 5, at least10, at least 20, at least 50, at least 100, at least 200, at least 500,at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, at least 100,000, at least 200,000, atleast 500,000, at least 1,000,000, at least 2,000,000, at least5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, or at least 1,000,000,000 cycles to process the analyte. Insome instances, a different solution may be directed to the substratefor each cycle. For example, at least 1, at least 2, at least 5, atleast 10, at least 20, at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, at least 100,000, at least 200,000, atleast 500,000, at least 1,000,000, at least 2,000,000, at least5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, or at least 1,000,000,000 solutions may be directed to thesubstrate.

In some instances, a washing solution may be directed to the substratebetween each cycle (or at least once during each cycle). For instance, awashing solution may be directed to the substrate after each type ofreaction mixture solution is directed to the substrate. The washingsolutions may be distinct. The washing solutions may be identical. Thewashing solution may be dispensed in pulses during rotation, creatingannular waves as described herein. For example, at least 1, at least 2,at least 5, at least 10, at least 20, at least 50, at least 100, atleast 200, at least 500, at least 1,000, at least 2,000, at least 5,000,at least 10,000, at least 20,000, at least 50,000, at least 100,000, atleast 200,000, at least 500,000, at least 1,000,000, at least 2,000,000,at least 5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, or at least 1,000,000,000 washing solutions may be directedto the substrate.

In some instances, a subset or an entirety of the solution(s) may berecycled after the solution(s) have contacted the substrate. Recyclingmay comprise collecting, filtering, and reusing the subset or entiretyof the solution. The filtering may be molecule filtering.

Nucleic Acid Sequencing Using a Rotating Array

In some instances, a method for sequencing may employ sequencing bysynthesis schemes wherein a nucleic acid molecule is sequencedbase-by-base with primer extension reactions. For example, a method forsequencing a nucleic acid molecule may comprise providing a substratecomprising an array having immobilized thereto the nucleic acidmolecule. The array may be a planar array. The substrate may beconfigured to rotate with respect to an axis. The method may comprisedirecting a solution comprising a plurality of nucleotides across thearray prior to or during rotation of the substrate. Rotation of thesubstrate may facilitate coating of the substrate surface with thesolution. The nucleic acid molecule may be subjected to a primerextension reaction under conditions sufficient to incorporate orspecifically bind at least one nucleotide from the plurality ofnucleotides into a growing strand that is complementary to the nucleicacid molecule. A signal indicative of incorporation or binding of atleast one nucleotide may be detected, thereby sequencing the nucleicacid molecule.

In some instances, the method may comprise, prior to providing thesubstrate having immobilized thereto the nucleic acid molecule,immobilizing the nucleic acid molecule to the substrate. For example, asolution comprising a plurality of nucleic acid molecules comprising thenucleic acid molecule may be directed to the substrate prior to, during,or subsequent to rotation of the substrate, and the substrate may besubject to conditions sufficient to immobilize at least a subset of theplurality of nucleic acid molecules as an array on the substrate.

FIG. 2 shows a flowchart for an example of a method 200 for sequencing anucleic acid molecule. In a first operation 210, the method may compriseproviding a substrate, as described elsewhere herein. The substrate maycomprise an array of a plurality of individually addressable locations.The array may be a planar array. The array may be a textured array. Thearray may be a patterned array. For example, the array may defineindividually addressable locations with wells and/or pillars. Aplurality of nucleic acid molecules, which may or may not be copies ofthe same nucleic acid molecule, may be immobilized to the array. Eachnucleic acid molecule from the plurality of nucleic acid molecules maybe immobilized to the array at a given individually addressable locationof the plurality of individually addressable locations.

The substrate may be configured to rotate with respect to an axis. Theaxis may be an axis through the center or substantially center of thesubstrate. The axis may be an off-center axis. For instance, thesubstrate may be affixed to a chuck (such as a vacuum chuck) of a spincoating apparatus. The substrate may be configured to rotate with arotational velocity of at least 1 revolution per minute (rpm), at least2 rpm, at least 5 rpm, at least 10 rpm, at least 20 rpm, at least 50rpm, at least 100 rpm, at least 200 rpm, at least 500 rpm, at least1,000 rpm, at least 2,000 rpm, at least 5,000 rpm, or at least 10,000rpm. The substrate may be configured to rotate with a rotationalvelocity that is within a range defined by any two of the precedingvalues. The substrate may be configured to rotate with differentrotational velocities during different operations described herein. Thesubstrate may be configured to rotate with a rotational velocity thatvaries according to a time-dependent function, such as a ramp, sinusoid,pulse, or other function or combination of functions. The time-varyingfunction may be periodic or aperiodic.

In a second operation 220, the method may comprise directing a solutionacross the array prior to or during rotation of the substrate. Thesolution may be centrifugally directed across the array. In someinstances, the solution may be directed to the array during rotation ofthe substrate in pulses, thereby creating an annular wave of thesolution moving radially outward. The solution may have a temperaturedifferent than that of the substrate, thereby providing a source or sinkof thermal energy to the substrate or to a nucleic acid molecule locatedon the substrate. The thermal energy may provide a temperature change tothe substrate or to the nucleic acid molecule. The temperature changemay be transient. The temperature change may enable, disable, enhance,or inhibit a chemical reaction, such as a chemical reaction to becarried out upon the nucleic acid molecule. The chemical reaction maycomprise denaturation, hybridization, or annealing of the plurality ofnucleic acid molecules. The chemical reaction may comprise a step in apolymerase chain reaction (PCR), bridge amplification, or other nucleicacid amplification reaction. The temperature change may modulate,increase, or decrease a signal detected from the nucleic acid molecules(or from probes in the solution).

In some instances, the solution may comprise probes configured tointeract with nucleic acid molecules. For example, in some instances,such as for performing sequencing by synthesis, the solution maycomprise a plurality of nucleotides (in single bases). The plurality ofnucleotides may include nucleotide analogs, naturally occurringnucleotides, and/or non-naturally occurring nucleotides, collectivelyreferred to herein as “nucleotides.” The plurality of nucleotides may ormay not be bases of the same type (e.g., A, T, G, C, etc.). For example,the solution may or may not comprise bases of only one type. Thesolution may comprise at least 1 type of base or bases of at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 types. For instance, the solution may compriseany possible mixture of A, T, C, and G. In some instances, the solutionmay comprise a plurality of natural nucleotides and non-naturalnucleotides. The plurality of natural nucleotides and non-naturalnucleotides may or may not be bases of the same type (e.g., A, T, G, C).In some cases, the solution may comprise probes that are oligomeric(e.g., oligonucleotide primers). For example, in some instances, such asfor performing sequencing by synthesis, the solution may comprise aplurality of nucleic acid molecules, e.g., primers, that comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotide bases. The plurality of nucleic acid molecules may comprisenucleotide analogs, naturally occurring nucleotides, and/ornon-naturally occurring nucleotides, collectively referred to herein as“nucleotides.” The plurality of nucleotides may or may not be bases ofthe same type (e.g., A, T, G, C, etc.). For example, the solution may ormay not comprise bases of only one type. The solution may comprise atleast 1 type of base or bases of at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, or at least 10types. For instance, the solution may comprise any possible mixture ofA, T, C, and G. In some instances, the solution may comprise a pluralityof natural nucleotides and non-natural nucleotides. The plurality ofnatural nucleotides and non-natural nucleotides may or may not be basesof the same type (e.g., A, T, G, C).

One or more nucleotides of the plurality of nucleotides may beterminated (e.g., reversibly terminated). For example, a nucleotide maycomprise a reversible terminator, or a moiety that is capable ofterminating primer extension reversibly. Nucleotides comprisingreversible terminators may be accepted by polymerases and incorporatedinto growing nucleic acid sequences analogously to non-reversiblyterminated nucleotides. A polymerase may be any naturally occurring(i.e., native or wild-type) or engineered variant of a polymerase (e.g.,DNA polymerase, Taq polymerase, etc.). Following incorporation of anucleotide analog comprising a reversible terminator into a nucleic acidstrand, the reversible terminator may be removed to permit furtherextension of the nucleic acid strand. A reversible terminator maycomprise a blocking or capping group that is attached to the 3′-oxygenatom of a sugar moiety (e.g., a pentose) of a nucleotide or nucleotideanalog. Such moieties are referred to as 3′-O-blocked reversibleterminators. Examples of 3′-O-blocked reversible terminators include,for example, 3′-ONH₂ reversible terminators, 3′-O-allyl reversibleterminators, and 3′-O-aziomethyl reversible terminators. Alternatively,a reversible terminator may comprise a blocking group in a linker (e.g.,a cleavable linker) and/or dye moiety of a nucleotide analog.3′-unblocked reversible terminators may be attached to both the base ofthe nucleotide analog as well as a fluorescing group (e.g., label, asdescribed herein). Examples of 3′-unblocked reversible terminatorsinclude, for example, the “virtual terminator” developed by HelicosBioSciences Corp. and the “lightning terminator” developed by Michael L.Metzker et al. Cleavage of a reversible terminator may be achieved by,for example, irradiating a nucleic acid molecule including thereversible terminator.

One or more nucleotides of the plurality of nucleotides may be labeledwith a dye, fluorophore, or quantum dot. For example, the solution maycomprise labeled nucleotides. In another example, the solution maycomprise unlabeled nucleotides. In another example, the solution maycomprise a mixture of labeled and unlabeled nucleotides. Non-limitingexamples of dyes include SYBR green, SYBR blue, DAPI, propidium iodine,Hoechst, SYBR gold, ethidium bromide, acridine, proflavine, acridineorange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, phenanthridines and acridines, ethidiumbromide, propidium iodide, hexidium iodide, dihydroethidium, ethidiumhomodimer-1 and -2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D,LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange,POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1,BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3,TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen,RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40,-41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11,-20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85(orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein,fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate(TRITC), rhodamine, tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3,Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC),Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD,ethidium homodimer I, ethidium homodimer II, ethidium homodimer III,ethidium bromide, umbelliferone, eosin, green fluorescent protein,erythrosin, coumarin, methyl coumarin, pyrene, malachite green,stilbene, lucifer yellow, cascade blue, dichlorotriazinylaminefluorescein, dansyl chloride, fluorescent lanthanide complexes such asthose including europium and terbium, carboxy tetrachloro fluorescein, 5and/or 6-carboxy fluorescein (FAM), VIC, 5- (or 6-)iodoacetamidofluorescein, 5-{[2(and3)-5-(Acetylmercapto)-succinyl]amino} fluorescein (SAMSA-fluorescein),lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine(ROX), 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid(AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acidtrisodium salt, 3,6-Disulfonate-4-amino-naphthalimide,phycobiliproteins, Atto 390, 425, 465, 488, 495, 532, 565, 594, 633,647, 647N, 665, 680 and 700 dyes, AlexaFluor 350, 405, 430, 488, 532,546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes,or other fluorophores, Black Hole Quencher Dyes (Biosearch Technologies)such as BHI-0, BHQ-1, BHQ-3, BHQ-10); QSY Dye fluorescent quenchers(from Molecular Probes/Invitrogen) such QSY7, QSY9, QSY21, QSY35, andother quenchers such as Dabcyl and Dabsyl; CySQ and Cy7Q and DarkCyanine dyes (GE Healthcare); Dy-Quenchers (Dyomics), such as DYQ-660and DYQ-661; and ATTO fluorescent quenchers (ATTO-TEC GmbH), such asATTO 540Q, 580Q, 612Q. In some cases, the label may be one with linkers.For instance, a label may have a disulfide linker attached to the label.Non-limiting examples of such labels include Cy5-azide, Cy-2-azide,Cy-3-azide, Cy-3.5-azide, Cy5.5-azide and Cy-7-azide. In some cases, alinker may be a cleavable linker. In some cases, the label may be a typethat does not self-quench or exhibit proximity quenching. Non-limitingexamples of a label type that does not self-quench or exhibit proximityquenching include Bimane derivatives such as Monobromobimane.Alternatively, the label may be a type that self-quenches or exhibitsproximity quenching. Non-limiting examples of such labels includeCy5-azide, Cy-2-azide, Cy-3-azide, Cy-3.5-azide, Cy5.5-azide andCy-7-azide. In some instances, a blocking group of a reversibleterminator may comprise the dye.

The solution may be directed to the array using one or more nozzles. Insome cases, different reagents (e.g., nucleotide solutions of differenttypes, washing solutions, etc.) may be dispensed via different nozzles,such as to prevent contamination. Each nozzle may be connected to adedicated fluidic line or fluidic valve, which may further preventcontamination. A type of reagent may be dispensed via one or morenozzles. The one or more nozzles may be directed at or in proximity to acenter of the substrate. Alternatively, the one or more nozzles may bedirected at or in proximity to a location on the substrate other thanthe center of the substrate. Two or more nozzles may be operated incombination to deliver fluids to the substrate more efficiently.

The solution may be dispensed on the substrate while the substrate isstationary; the substrate may then be subjected to rotation followingthe dispensing of the solution. Alternatively, the substrate may besubjected to rotation prior to the dispensing of the solution; thesolution may then be dispensed on the substrate while the substrate isrotating. Rotation of the substrate may yield a centrifugal force (orinertial force directed away from the axis) on the solution, causing thesolution to flow radially outward over the array.

In a third operation 230, the method may comprise subjecting the nucleicacid molecule to a primer extension reaction. The primer extensionreaction may be conducted under conditions sufficient to incorporate atleast one nucleotide from the plurality of nucleotides into a growingstrand that is complementary to the nucleic acid molecule. Thenucleotide incorporated may or may not be labeled.

In some cases, the operation 230 may further comprise modifying at leastone nucleotide. Modifying the nucleotide may comprise labeling thenucleotide. For instance, the nucleotide may be labeled, such as with adye, fluorophore, or quantum dot. The nucleotide may be cleavablylabeled. In some instances, modifying the nucleotide may compriseactivating (e.g., stimulating) a label of the nucleotide.

In a fourth operation 240, the method may comprise detecting a signalindicative of incorporation of the at least one nucleotide. The signalmay be an optical signal. The signal may be a fluorescence signal. Thesignal may be detected during rotation of the substrate. The signal maybe detected following termination of the rotation. The signal may bedetected while the nucleic acid molecule to be sequenced is in fluidcontact with the solution. The signal may be detected following fluidcontact of the nucleic acid molecule with the solution. The operation240 may further comprise modifying a label of the at least onenucleotide. For instance, the operation 240 may further comprisecleaving the label of the nucleotide (e.g., after detection). Thenucleotide may be cleaved by one or more stimuli, such as exposure to achemical, an enzyme, light (e.g., ultraviolet light), or heat. Once thelabel is cleaved, a signal indicative of the incorporated nucleotide maynot be detectable with one or more detectors.

The method 200 may further comprise repeating operations 220, 230,and/or 240 one or more times to identify one or more additional signalsindicative of incorporation of one or more additional nucleotides,thereby sequencing the nucleic acid molecule. The method 200 maycomprise repeating operations 220, 230, and/or 240 in an iterativemanner. For each iteration, an additional signal may indicateincorporation of an additional nucleotide. The additional nucleotide maybe the same nucleotide as detected in the previous iteration. Theadditional nucleotide may be a different nucleotide from the nucleotidedetected in the previous iteration. In some instances, at least onenucleotide may be modified (e.g., labeled and/or cleaved) between eachiteration of the operations 220, 230, or 240. For instance, the methodmay comprise repeating the operations 220, 230, and/or 240 at least 1,at least 2, at least 5, at least 10, at least 20, at least 50, at least100, at least 200, at least 500, at least 1,000, at least 2,000, atleast 5,000, at least 10,000, at least 20,000, at least 50,000, at least100,000, at least 200,000, at least 500,000, at least 1,000,000, atleast 2,000,000, at least 5,000,000, at least 10,000,000, at least20,000,000, at least 50,000,000, at least 100,000,000, at least200,000,000, at least 500,000,000, or at least 1,000,000,000 times. Themethod may comprise repeating the operations 220, 230, and/or 240 anumber of times that is within a range defined by any two of thepreceding values. The method 200 may thus result in the sequencing of anucleic acid molecule of any size.

The method may comprise directing different solutions to the arrayduring rotation of the substrate in a cyclical manner. For instance, themethod may comprise directing a first solution containing a first typeof nucleotide (e.g., in a plurality of nucleotides of the first type) tothe array, followed by a second solution containing a second type ofnucleotide, followed by a third type of nucleotide, followed by a fourthtype of nucleotide, etc. In another example, different solutions maycomprise different combinations of types of nucleotides. For example, afirst solution may comprise a first canonical type of nucleotide (e.g.,A) and a second canonical type of nucleotide (e.g., C), and a secondsolution may comprise the first canonical type of nucleotide (e.g., A)and a third canonical type of nucleotide (e.g., T), and a third solutionmay comprise the first canonical type, second canonical type, thirdcanonical type, and a fourth canonical type (e.g., G) of nucleotide. Inanother example, a first solution may comprise labeled nucleotides, anda second solution may comprise unlabeled nucleotides, and a thirdsolution may comprise a mixture of labeled and unlabeled nucleotides.The method may comprise directing at least 1, at least 2, at least 5, atleast 10, at least 20, at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, at least 100,000, at least 200,000, atleast 500,000, at least 1,000,000, at least 2,000,000, at least5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, or at least 1,000,000,000 solutions to the array. Themethod may comprise directing a number of solutions that is within arange defined by any two of the preceding values to the array. Thesolutions may be distinct. The solutions may be identical.

The method may comprise directing at least 1, at least 2, at least 5, atleast 10, at least 20, at least 50, at least 100, at least 200, at least500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, atleast 20,000, at least 50,000, at least 100,000, at least 200,000, atleast 500,000, at least 1,000,000, at least 2,000,000, at least5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, or at least 1,000,000,000 washing solutions to thesubstrate. For instance, a washing solution may be directed to thesubstrate after each type of nucleotide is directed to the substrate.The washing solutions may be distinct. The washing solutions may beidentical. The washing solution may be dispensed in pulses duringrotation, creating annular waves as described herein.

The method may further comprise recycling a subset or an entirety of thesolution(s) after the solution(s) have contacted the substrate.Recycling may comprise collecting, filtering, and reusing the subset orentirety of the solution. The filtering may be molecule filtering.

The operations 220 and 230 may occur at a first location and theoperation 240 may occur at a second location. The first and secondlocations may comprise first and second processing bays, respectively,as described herein (for instance, with respect to FIG. 23H). The firstand second locations may comprise first and second rotating spindles,respectively, as described herein (for instance, with respect to FIG.24). The first rotating spindle may be exterior or interior to thesecond rotating spindle. The first and second rotating spindles may beconfigured to rotate with different angular velocities. Alternatively,the operation 220 may occur at a first location and the operations 230and 240 may occur at the second location.

The method may further comprise transferring the substrate between thefirst and second locations. Operations 220 and 230 may occur while thesubstrate is rotated at a first angular velocity and operation 240 mayoccur while the substrate is rotated at a second angular velocity. Thefirst angular velocity may be less than the second angular velocity. Thefirst angular velocity may be between about 0 rpm and about 100 rpm. Thesecond angular velocity may be between about 100 rpm and about 1,000rpm. Alternatively, the operation 220 may occur while the substrate isrotated at the first angular velocity and the operations 230 and 240 mayoccur while the substrate is rotated at the second angular velocity.

Many variations, alterations, and adaptations based on the method 200provided herein are possible. For example, the order of the operationsof the method 200 may be changed, some of the operations removed, someof the operations duplicated, and additional operations added asappropriate. Some of the operations may be performed in succession. Someof the operations may be performed in parallel. Some of the operationsmay be performed once. Some of the operations may be performed more thanonce. Some of the operations may comprise sub-operations. Some of theoperations may be automated. Some of the operations may be manual. Someof the operations may be performed separately, e.g., in differentlocations or during different steps and/or processes. For example,directing a solution comprising a plurality of probes to the substratemay occur separately from the reaction and detection processes.

For example, in some cases, in the third operation 230, instead offacilitating a primer extension reaction, the nucleic acid molecule maybe subject to conditions to allow transient binding of a nucleotide fromthe plurality of nucleotides to the nucleic acid molecule. Thetransiently bound nucleotide may be labeled. The transiently boundnucleotide may be removed, such as after detection (e.g., see operation240). Then, a second solution may be directed to the substrate, thistime under conditions to facilitate the primer extension reaction, suchthat a nucleotide of the second solution is incorporated (e.g., into agrowing strand hybridized to the nucleic acid molecule). Theincorporated nucleotide may be unlabeled. After washing, and withoutdetecting, another solution of labeled nucleotides may be directed tothe substrate, such as for another cycle of transient binding.

In some instances, such as for performing sequencing by ligation, thesolution may comprise different probes. For example, the solution maycomprise a plurality of oligonucleotide molecules. For example, theoligonucleotide molecules may have a length of about 2 bases, 3 bases, 4bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases or more.The oligonucleotide molecules may be labeled with a dye (e.g.,fluorescent dye), as described elsewhere herein. In some instances, suchas for detecting repeated sequences in nucleic acid molecules, such ashomopolymer repeated sequences, dinucleotide repeated sequences, andtrinucleotide repeated sequences, the solution may comprise targetedprobes (e.g., homopolymer probe) configured to bind to the repeatedsequences. The solution may comprise one type of probe (e.g.,nucleotides). The solution may comprise different types of probes (e.g.,nucleotides, oligonucleotide molecules, etc.). The solution may comprisedifferent types of probes (e.g., oligonucleotide molecules, antibodies,etc.) for interacting with different types of analytes (e.g., nucleicacid molecules, proteins, etc.). Different solutions comprisingdifferent types of probes may be directed to the substrate any number oftimes, with or without detection between consecutive cycles (e.g.,detection may be performed between some consecutive cycles, but notbetween some others), to sequence or otherwise process the nucleic acidmolecule, depending on the type of processing.

FIG. 3 shows a system 300 for sequencing a nucleic acid molecule orprocessing an analyte. The system may be configured to implement themethod 200 or 1400. Although the systems (e.g., 300, 400, 500 a, 500 b,etc.) are described with respect to processing nucleic acid molecules,the systems may be used to process any other type of biological analyte,as described herein.

The system may comprise a substrate 310. The substrate may comprise anysubstrate described herein, such as any substrate described herein withrespect to FIG. 2. The substrate may comprise an array. The substratemay be open. The array may comprise one or more locations 320 configuredto immobilize one or more nucleic acid molecules or analytes. The arraymay comprise any array described herein, such as any array describedherein with respect to method 200. For instance, the array may comprisea plurality of individually addressable locations. The array maycomprise a linker (e.g., any binder described herein) that is coupled tothe nucleic acid molecule to be sequenced. Alternatively or incombination, the nucleic acid molecule to be sequenced may be coupled toa bead; the bead may be immobilized to the array. The array may betextured. The array may be a patterned array. The array may be planar.

The substrate may be configured to rotate with respect to an axis 305.The axis may be an axis through the center of the substrate. The axismay be an off-center axis. The substrate may be configured to rotate atany rotational velocity described herein, such as any rotationalvelocity described herein with respect to method 200 or 1400.

The substrate may be configured to undergo a change in relative positionwith respect to first or second longitudinal axes 315 and 325. Forinstance, the substrate may be translatable along the first and/orsecond longitudinal axes (as shown in FIG. 3). Alternatively, thesubstrate may be stationary along the first and/or second longitudinalaxes. Alternatively or in combination, the substrate may be translatablealong the axis (as shown in FIG. 4). Alternatively or in combination,the substrate may be stationary along the axis. The relative position ofthe substrate may be configured to alternate between positions. Therelative position of the substrate may be configured to alternatebetween positions with respect to one or more of the longitudinal axesor the axis. The relative position of the substrate may be configured toalternate between positions with respect to any of the fluid channelsdescribed herein. For instance, the relative position of the substratemay be configured to alternate between a first position and a secondposition. The relative position of the substrate may be configured toalternate between at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 positions. Therelative position of the substrate may be configured to alternatebetween a number of positions that is within a range defined by any twoof the preceding values. The first or second longitudinal axes may besubstantially perpendicular with the axis. The first or secondlongitudinal axes may be substantially parallel with the axis. The firstor second longitudinal axes may be coincident with the axis.

The system may comprise a first fluid channel 330. The first fluidchannel may comprise a first fluid outlet port 335. The first fluidoutlet port may be configured to dispense a first fluid to the array.The first fluid outlet port may be configured to dispense any fluiddescribed herein, such as any solution described herein. The first fluidoutlet port may be external to the substrate. The first fluid outletport may not contact the substrate. The first fluid outlet port may be anozzle. The first fluid outlet port may have an axis that issubstantially coincident with the axis. The first fluid outlet port mayhave an axis that is substantially parallel to the axis.

The system may comprise a second fluid channel 340. The second fluidchannel may comprise a second fluid outlet port 345. The second fluidoutlet port may be configured to dispense a second fluid to the array.The second fluid outlet port may be configured to dispense any fluiddescribed herein, such as any solution described herein. The secondfluid outlet port may be external to the substrate. The second fluidoutlet port may not contact the substrate. The second fluid outlet portmay be a nozzle. The second fluid outlet port may have an axis that issubstantially coincident with the axis. The second fluid outlet port mayhave an axis that is substantially parallel to the axis.

The first and second fluids may comprise different types of reagents.For instance, the first fluid may comprise a first type of nucleotide,such as any nucleotide described herein, or a nucleotide mixture. Thesecond fluid may comprise a second type of nucleotide, such as anynucleotide described herein, or a nucleotide mixture. Alternatively, thefirst and second fluids may comprise the same type of reagents (e.g.,same type of fluid is dispensed through multiple fluid outlet ports(e.g., nozzles) to increase coating speed). Alternatively or incombination, the first or second fluid may comprise a washing reagent.The first fluid channel 330 and the second fluid channel 340 may befluidically isolated. Beneficially, where the first and second fluidscomprise different types of reagents, each of the different reagents mayremain free of contamination from the other reagents during dispensing.

The first fluid outlet port may be configured to dispense the firstfluid during rotation of the substrate. The second fluid outlet port maybe configured to dispense the second fluid during rotation of thesubstrate. The first and second fluid outlet ports may be configured todispense at non-overlapping times. Alternatively, the first and secondfluid outlet ports may be configured to dispense at overlapping times,such as when the first fluid and the second fluid comprise the same typeof reagents. The substrate may be configured to rotate with a differentspeed or a different number of rotations when the first and secondoutlet ports dispense. Alternatively, the substrate may be configured torotate with the same speed and number of rotations when the first andsecond outlet ports dispense. During rotation, the array may beconfigured to direct the first fluid in a substantially radial directionaway from the axis. The first fluid outlet port may be configured todirect the first fluid to the array during at least 1, at least 2, atleast 5, at least 10, at least 20, at least 50, at least 100, at least200, at least 500, at least 1,000, at least 2,000, at least 5,000, atleast 10,000, at least 20,000, at least 50,000, at least 100,000, atleast 200,000, at least 500,000, or at least 1,000,000 full rotations ofthe substrate. The first fluid outlet port may be configured to directthe first fluid to the array during a number of full rotations that iswithin a range defined by any two of the preceding values.

The system may comprise a third fluid channel 350 comprising a thirdfluid outlet port 355 configured to dispense a third fluid. The systemmay comprise a fourth fluid channel 360 comprising a fourth fluid outletport 365 configured to dispense a fourth fluid. The third and fourthfluid channels may be similar to the first and second fluid channelsdescribed herein. The third and fourth fluids may be the same ordifferent fluids as the first and/or second fluids. In some cases, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more fluids (or reagents)may be employed. For example, 5-10 fluids (or reagents) may be employed.

Although FIG. 3 shows a change in position of the substrate, as analternative or in addition to, one or more of the first, second, third,and fourth fluid channels may be configured to undergo a change inposition. For instance, any of the first, second, third, or fourth fluidchannel may be translatable along the first and/or second longitudinalaxes. Alternatively, any of the first, second, third, or fourth fluidchannel may be stationary along the first and/or second longitudinalaxes. Alternatively or in addition, any of the first, second, third, orfourth fluid channel may be translatable along the axis. Alternativelyor in addition, any of the first, second, third, or fourth fluid channelmay be stationary along the axis.

The relative position of one or more of the first, second, third, andfourth fluid channels may be configured to alternate between positionswith respect to one or more of the longitudinal axes or the axis. Forinstance, the relative position of any of the first, second, third, orfourth fluid channel may be configured to alternate between a firstposition and a second position (e.g., by moving such channel, by movingthe substrate, or by moving the channel and the substrate). The relativeposition of any of the first, second, third, or fourth fluid channel maybe configured to alternate between at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20 or more positions. The relative position of any of the first, second,third, or fourth fluid channel may be configured to alternate between anumber of positions that is within a range defined by any two of thepreceding values. The first or second longitudinal axes may besubstantially perpendicular to the axis. The first or secondlongitudinal axes may be substantially parallel to the axis. The firstor second longitudinal axes may be coincident with the axis.

In some instances, the system may comprise one or more fluid channelsfor receiving fluid from the substrate (not shown in FIG. 3). Referringto FIG. 4A-FIG. 4B, a fifth fluid channel 430 may comprise a first fluidinlet port 435. The first fluid inlet port may be located at a firstlevel of the axis (as shown in FIG. 4). In some instances, the firstfluid inlet port may surround the periphery of the substrate 310 (e.g.,circularly). The first fluid inlet port may be downstream of and influid communication with the substrate 310 when the substrate is in afirst position, such as with respect to the axis. The fifth fluidchannel may be in fluid communication with the first fluid channel 330(as shown in FIG. 3). For example, the first fluid inlet port may beconfigured to receive a solution passing from the first fluid outletport to the substrate and thereafter off the substrate (e.g., due toinertial forces during rotation of the substrate). For instance, thefirst fluid inlet port may be configured to receive the solution in arecycling process such as the recycling process described herein withrespect to method 200 or 1400. In some instances, the solution receivedby the fifth fluid channel via the first fluid inlet port may be fedback (e.g., after filtering) to the first fluid channel to be dispensedvia the first fluid outlet port to the substrate. The fifth fluidchannel and the first fluid channel may define at least part of a firstcyclic fluid flow path. The first cyclic fluid flow path may comprise afilter, such as a filter described herein with respect to method 200 or1400. The filter may be a molecular filter. In other instances, thesolution received by the fifth fluid channel may be fed back (e.g.,after filtering) to different fluid channels (other than the first fluidchannel) to be dispensed via different fluid outlet ports.

The system may comprise a sixth fluid channel 440. The sixth fluidchannel may comprise a second fluid inlet port 445. The second fluidinlet port may be located at a second level of the axis (as shown inFIG. 4). In some instances, the second fluid inlet port may surround theperiphery of the substrate 310. The second fluid inlet port may bedownstream of and in fluid communication with the substrate 310 when thesubstrate is in a second position, such as with respect to the axis. Thesixth fluid channel may be in fluid communication with the second fluidchannel 340. For example, the second fluid inlet port may be configuredto receive a solution passing from the second fluid outlet port to thesubstrate and thereafter off the substrate. For instance, the secondfluid inlet port may be configured to receive the solution in arecycling process such as the recycling process described herein withrespect to method 200 or 1400. In some instances, the solution receivedby the sixth fluid channel via the second fluid inlet port may be fedback (e.g., after filtering) to the second fluid channel to be dispensedvia the second fluid outlet port to the substrate. The sixth fluidchannel and the second fluid channel may define at least part of asecond cyclic fluid flow path. The second cyclic fluid flow path maycomprise a filter, such as a filter described herein with respect tomethod 200 or 1400. The filter may be a molecular filter.

The system may comprise a shield (not shown) that prevents fluidcommunication between the substrate and the second fluid inlet port whenthe substrate is in the first position and between the substrate and thefirst fluid inlet port when the substrate is in the second position.

The system may further comprise one or more detectors 370. The detectorsmay be optical detectors, such as one or more photodetectors, one ormore photodiodes, one or more avalanche photodiodes, one or morephotomultipliers, one or more photodiode arrays, one or more avalanchephotodiode arrays, one or more cameras, one or more charged coupleddevice (CCD) cameras, or one or more complementary metal oxidesemiconductor (CMOS) cameras. The cameras may be TDI or other continuousarea scanning detectors described herein, including, for example, TDIline-scan cameras. The detectors may be fluorescence detectors. Thedetectors may be in sensing communication with the array. For instance,the detectors may be configured to detect a signal from the array. Thesignal may be an optical signal. The signal may be a fluorescencesignal. The detectors may be configured to detect the signal from thesubstrate during rotation of the substrate. The detectors may beconfigured to detect the signal from the substrate when the substrate isnot rotating. The detectors may be configured to detect the signal fromthe substrate following termination of the rotation of the substrate.FIG. 3 shows an example region 375 on the substrate that is opticallymapped to the detector.

The system may comprise one or more sources (not shown in FIG. 3)configured to deliver electromagnetic radiation to the substrate. Thesources may comprise one or more optical sources (e.g., illuminationsources). The sources may comprise one or more incoherent or coherentoptical sources. The sources may comprise one or more narrow bandwidthor broadband optical sources. The sources may be configured to emitoptical radiation having a bandwidth of at most 1 hertz (Hz), at most 2Hz, at most 5 Hz, at most 10 Hz, at most 20 Hz, at most 50 Hz, at most100 Hz, at most 200 Hz, at most 500 Hz, at most 1 kilohertz (kHz), atmost 2 kHz, at most 5 kHz, at most 10 kHz, at most 20 kHz, at most 50kHz, at most 100 kHz, at most 200 kHz, at most 500 kHz, at most 1megahertz (MHz), at most 2 MHz, at most 5 MHz, at most 10 MHz, at most20 MHz, at most 50 MHz, at most 100 MHz, at most 200 MHz, at most 500MHz, at most 1 gigahertz (GHz), at most 2 GHz, at most 5 GHz, at most 10GHz, at most 20 GHz, at most 50 GHz, at most 100 GHz, or a bandwidththat is within a range defined by any two of the preceding values. Thesource may comprise one or more light emitting diodes (LEDs). Thesources may comprise one or more lasers. The sources may comprise one ormore single-mode laser sources. The sources may comprise one or moremulti-mode laser sources. The sources may comprise one or more laserdiodes. A laser may be a continuous wave laser or a pulsed laser. A beamof light emitted by a laser may be a Gaussian or approximately Gaussianbeam, which beam may be manipulated using one or more optical elements(e.g., mirrors, lenses, prisms, waveplates, etc.). For example, a beammay be collimated. In some cases, a beam may be manipulated to provide alaser line (e.g., using one or more Powell lenses or cylindricallenses). FIG. 11A shows an example of beam shaping using a cylindricallens to provide a laser line. A collimated beam having a radius r₀ isincident upon a cylindrical plano-concave lens having a focal length −f.The beam will expand with a half-angle θ equivalent to r₀/f. The laserline will have a thickness of approximately 2r₀ and a length L ofapproximately 2(r₀/f)(z+f) at a distance z from the lens. In someembodiments, a beam thickness may be expanded along a single axis, forexample the y-axis, while the beam thickness remains substantiallyunchanged along a second axis, for example the x-axis, as shown in FIG.11B. Expansion along a single axis may be achieved using a cylindricallens, for example a plano-concave cylindrical lens having a focal lengthof −f along the axis of expansion. The beam shaping lens may be part ofa line shaper element, as shown in FIG. 11C. The line shaper element maycomprise one or more optical elements configured to expand a beam alonga single axis. The line shaper element may further comprise one or moreoptical elements to collimate the expanded beam, for example a secondcylindrical lens. In some embodiments, the second cylindrical lens is aplano-convex cylindrical lens. The expanded beam may result in a laserline, as shown in FIG. 11B. A laser line may impinge directly on asubstrate or may be projected onto the substrate such that isapproximately perpendicular to a central axis about which the opensubstrate may rotate.

The sources (e.g., optical or illumination sources) of a system may beconfigured to emit light comprising one or more wavelengths in theultraviolet (about 100 nm to about 400 nm), visible (about 400 nm toabout 700 nm), or infrared (about 700 nm to about 10,000 nm) regions ofthe electromagnetic spectrum, or any combination therefore. Forinstances, the sources may emit radiation comprising one or morewavelengths in the range from 600 nm to 700 nm. The sources may emitradiation, either individually or in combination, having an opticalpower of at least 0.05 watts (W), at least 0.1 W, at least 0.2 W, atleast 0.5 W, at least 1 W, at least 2 W, at least 5 W, at least 10 W, oran optical power that is within a range defined by any two of thepreceding values. The sources may be configured to interact withmolecules on the substrate to generate detectable optical signals thatmay be detected by the optical detectors. For instance, the sources maybe configured to generate optical absorption, optical reflectance,scattering, phosphorescence, fluorescence, or any other optical signaldescribed herein.

The system may comprise a seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth,eighteenth, nineteenth, or twentieth fluid channel. Each fluid channelmay comprise a fluid outlet port or a fluid inlet port in fluidcommunication with the substrate. For instance, the ninth, tenth,thirteenth, fourteenth, seventeenth, or eighteenth fluid channel maycomprise a fluid outlet port. The seventh, eighth, eleventh, twelfth,fifteenth, sixteenth, nineteenth, or twentieth fluid channel maycomprise a fluid inlet port. Alternatively, the system may comprise morethan twenty fluid channels comprising a fluid outlet port or a fluidinlet port.

Thus, the system may comprise fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports. The fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports may be configured to dispense fifth, sixth,seventh, eighth, ninth, or tenth fluids to the array. The fifth, sixth,seventh, eighth, ninth, or tenth fluid outlet ports may be configured todispense any fluid described herein, such as any solution describedherein. The fifth, sixth, seventh, eighth, ninth, or tenth fluid outletports may be similar to the first, second, third, or fourth fluid outletports described herein. Alternatively, the system may comprise more thanten fluid outlet ports.

The fluid channels may be fluidically isolated from one another. Forinstance, the fluid channels may be fluidically isolated upstream of thefirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports. The fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports may be external to the substrate. The fifth,sixth, seventh, eighth, ninth, or tenth fluid outlet ports may notcontact the substrate. The fifth, sixth, seventh, eighth, ninth, ortenth fluid outlet ports may be a nozzle.

The system may comprise third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth fluid inlet ports. The third, fourth, fifth, sixth,seventh, eighth, ninth, or tenth fluid inlet ports may be in fluidcommunication with the substrate when the substrate is in a third,fourth, fifth, sixth, seventh, eighth, ninth, or tenth position (e.g.,with respect to the axis), respectively. Alternatively, the system maycomprise more than ten fluid inlet ports.

The ninth, tenth, thirteenth, fourteenth, seventeenth, or eighteenthfluid channel may be in fluid communication with the seventh, eighth,eleventh, twelfth, fifteenth, or sixteenth, fluid channel, respectively;each pair of fluid channels may define at least part of a third, fourth,fifth, sixth, seventh, eighth, ninth, or tenth cyclic fluid flow path,respectively. Each cyclic fluid flow path may be configured similarly tothe first or second cyclic fluid flow paths described herein, with thefluid inlet port of the cyclic fluid flow path configured to receive asolution passing from the fluid outlet port of the cyclic fluid flowpath to the substrate. Each cyclic fluid flow path may be configured toreceive the solution in a recycling process as described herein. Eachcyclic fluid flow path may comprise a filter as described herein.

The fifth, sixth, seventh, eighth, ninth, or tenth fluids may comprisedifferent types of reagents. For instance, the fifth, sixth, seventh,eighth, ninth, or tenth fluid may comprise a fifth, sixth, seventh,eighth, ninth, or tenth type of nucleotide, respectively, such as anynucleotide described herein. Alternatively or in combination, the fifth,sixth, seventh, eighth, ninth, or tenth fluid may comprise a washingreagent.

The fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port maybe configured to dispense the fifth, sixth, seventh, eighth, ninth, ortenth fluid, respectively, during rotation of the substrate. The fifth,sixth, seventh, eighth, ninth, or tenth fluid outlet ports may beconfigured to dispense at overlapping or non-overlapping times.

FIG. 4A shows a system 400 for sequencing a nucleic acid molecule in afirst vertical level. The system may be substantially similar to system300 described herein or may differ from system 300 in the arrangement ofone or more of its elements. The system 400 may comprise substrate 310described herein. The system 400 may utilize vertical motion parallel tothe axis 305 to expose (e.g., make available fluid communication) thesubstrate 310 to different fluid channels. The system may comprise firstfluid channel 330 and first fluid outlet port 335 described herein. Thesystem may comprise second fluid channel 340 and second fluid outletport 345 described herein. The system may comprise third fluid channel350 and third fluid outlet port 355 described herein. The system maycomprise fourth fluid channel 360 and fourth fluid outlet port 365described herein. The system may comprise detector 370 described herein.The detector may be in optical communication with the region shown. Thesystem may comprise any optical source described herein (not shown inFIG. 4A).

The fifth fluid channel 430 and first fluid inlet port 435 may bearranged at a first level along the vertical axis, as shown in FIG. 4Aand FIG. 4B. The sixth fluid channel 440 and second fluid inlet port 445may be arranged at a second level along the vertical axis. In thismanner, the system may be viewed as comprising first and second fluidflow paths, with each fluid flow path located at a different verticallevel. The substrate 310 may be vertically movable between the firstlevel and the second level, from the first level to the second level,and from the second level to the first level. As an alternative, thesubstrate may be vertically fixed, but the levels may be verticallymovable with respect to the substrate 310. As another alternative, thesubstrate and the levels may be vertically movable.

The system 400 may comprise multiple levels. The levels may bevertically orientated relative to one another. The system may include atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more levels.Each level may include one or more sub-levels (e.g., an incrementallevel between any two levels). Each level may be for dispensing and/orrecovering a different fluid (or reagent). Some levels may be fordispensing the same fluid (or reagent).

While in the first vertical level, the substrate may be in fluidcommunication with the fifth fluid channel and the first fluid inletport, but not the sixth fluid channel and the second fluid inlet port.The substrate may be isolated from the sixth fluid channel and thesecond fluid inlet port by a shield (not shown), as described herein. Afirst fluid or first solution described herein may be dispensed to thesubstrate while the substrate is in this first vertical level. Forexample, any excess of the first solution spinning off the substrate maybe received by the first fluid inlet port while the substrate is at thefirst vertical level. In another example, a washing solution (e.g.,dispensed from a different fluid outlet port than the first fluid)spinning off the substrate with some of the first fluid may be receivedby the first fluid inlet port while the substrate is at the firstvertical level. The substrate may then be moved to a second verticallevel by vertically moving the substrate. Alternatively, the fifth orsixth fluid channels may be moved vertically. Alternatively or inaddition, the substrate and one or more of the fluid channels may bemoved relative to the other (e.g., along the axis).

FIG. 4B shows the system 400 for sequencing a nucleic acid molecule in asecond vertical level. While in the second vertical level, the substratemay be in fluid communication with the sixth fluid channel and thesecond fluid inlet port, but not the fifth fluid channel and the firstfluid inlet port. The substrate may be isolated from the fifth fluidchannel and the first fluid inlet port by a shield (not shown), asdescribed herein. A second fluid or second solution described herein maybe dispensed to the substrate while the substrate is in this secondvertical position. Alternatively, the first solution may be removedwhile the substrate is in the second vertical position. In some cases,the first solution may be recycled while the substrate is in the secondvertical position. The substrate may then be moved back to the firstvertical level, or to another vertical level described herein, byvertically moving the substrate. Alternatively, the fifth or sixth fluidchannels may be moved vertically. Alternatively or in addition, thesubstrate and one or more of the fluid channels may be moved relative tothe other (e.g., along the axis).

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluidinlet ports may be located at third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth vertical levels, respectively. The substrate maybe moved to the third, fourth, fifth, sixth, seventh, eighth, ninth, ortenth vertical levels by vertically moving the substrate or byvertically moving the first, second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth,fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth,or twentieth fluid flow channels. At any of the first, second, third,fourth, fifth, sixth, seventh, eighth, ninth, tenth or more verticallevels, any fluid solution described herein may be dispensed to thesubstrate. At any of the first, second, third, fourth, fifth, sixth,seventh, eighth, ninth, tenth or more vertical levels, any fluidsolution described herein may be removed from the substrate. At any ofthe first, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth or more vertical levels, any fluid solution described herein maybe recycled from the substrate.

FIG. 5A shows a first example of a system 500 a for sequencing a nucleicacid molecule using an array of fluid flow channels. The system may besubstantially similar to system 300 or 400 described herein and maydiffer from system 300 or 400 in the arrangement of one or more of itselements. The system 500 a may utilize a geometrical arrangement of aplurality of fluid flow channels to expose the substrate to differentfluids. The system 500 a may comprise substrate 310 described herein.The system may comprise first fluid channel 330 and first fluid outletport 335 described herein. The system may comprise second fluid channel340 and second fluid outlet port 345 described herein. The system maycomprise fifth fluid channel 430 and first fluid inlet port 435described herein (not shown in FIG. 5A). The system may comprise sixthfluid channel 440 and second fluid inlet port 445 described herein (notshown in FIG. 5A). The system may comprise detector 370 described herein(not shown in FIG. 5A). The system may comprise any illumination sourcedescribed herein (not shown in FIG. 5A).

The first fluid channel and first fluid outlet port may be arranged at afirst position, as shown in FIG. 5A. The second fluid channel and secondfluid outlet port may be arranged at a second position. The system maybe configured to dispense a first fluid from the first fluid outlet portand a second fluid from the second fluid outlet port.

The system may comprise any of third, fourth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth,seventeenth, eighteenth, nineteenth, or twentieth fluid channelsdescribed herein. The system may comprise any of third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth fluid outlet ports describedherein. The system may comprise any of third, fourth, fifth, sixth,seventh, eighth, ninth, or tenth fluid inlet ports described herein.

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluidoutlet ports may be located at third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth positions, respectively. The system may beconfigured to dispense a third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth fluid from the third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth fluid outlet port, respectively.

Any two or more of the first, second, third, fourth, seventh, eighth,ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth,sixteenth, seventeenth, eighteenth, nineteenth, twentieth, or more fluidchannels may form an array of fluid flow channels. The array of fluidflow channels may be moveable. Alternatively, the array of fluid flowchannels may be at a fixed location with respect to the substrate. Eachfluid flow channel of the array of fluid flow channels may be positionedsuch that a longitudinal axis of the fluid flow channel forms an anglewith the rotational axis of the substrate. The angle may have a value ofat least 0 degrees, at least 5 degrees, at least 10 degrees, at least 15degrees, at least 20 degrees, at least 25 degrees, at least 30 degrees,at least 35 degrees, at least 40 degrees, at least 45 degrees, at least50 degrees, at least 55 degrees, at least 60 degrees, at least 65degrees, at least 70 degrees, at least 75 degrees, at least 80 degrees,at least 85 degrees, or at least 90 degrees. The angle may have a valuethat is within a range defined by any two of the preceding values. Eachfluid channel of the array of fluid channels may make a similar anglewith the substrate. Alternatively, one or more fluid channels may makedifferent angles with the substrate.

FIG. 5B shows a second example of a system 500 b for sequencing anucleic acid molecule using an array of fluid flow channels.

The system may be substantially similar to system 300 or 400 describedherein and may differ from system 300 or 400 in the arrangement of oneor more of its elements. The system 500 b may utilize a plurality offluid flow channels configured to move relative to the substrate toexpose the substrate to different fluids. The system 500 b may comprisesubstrate 310 described herein. The system may comprise first fluidchannel 330 and first fluid outlet port 335 described herein. The systemmay comprise second fluid channel 340 and second fluid outlet port 345described herein. The system may comprise fifth fluid channel 430 andfirst fluid inlet port 435 described herein (not shown in FIG. 5B). Thesystem may comprise sixth fluid channel 440 and second fluid inlet port445 described herein (not shown in FIG. 5B). The system may comprisedetector 370 described herein (not shown in FIG. 5B). The system maycomprise any optical source described herein (not shown in FIG. 5B).

The first fluid channel and first fluid outlet port may be attached to afluid dispenser 510. The fluid dispenser may be a moveable fluiddispenser, such as comprising a moveable gantry arm, as shown in FIG.5B. As an alternative, the fluid dispenser may be fixed or stationary.The fluid dispenser may be configured to move to a first position todispense a first fluid from the first fluid outlet port. The secondfluid channel and second fluid outlet port may also be attached to thefluid dispenser. The fluid dispenser may be configured to move to asecond position to dispense a second fluid from the second fluid outletport.

The system may comprise any of third, fourth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth,seventeenth, eighteenth, nineteenth, or twentieth fluid channelsdescribed herein. The system may comprise any of third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth fluid outlet ports describedherein. The system may comprise any of third, fourth, fifth, sixth,seventh, eighth, ninth, or tenth fluid inlet ports described herein.

The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth fluidoutlet ports may be attached to the fluid dispenser. The fluid dispensermay be configured to move to a third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth position to dispense a third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth fluid from the third, fourth,fifth, sixth, seventh, eighth, ninth, or tenth fluid outlet port,respectively. Alternatively, the fluid dispenser may be kept stationaryand the substrate 310 may be moved to different positions to receivedifferent fluids.

FIG. 6 shows a computerized system 600 for sequencing a nucleic acidmolecule. The system may comprise a substrate 310, such as a substratedescribed herein with respect to method 200 or 1400, or system 300. Thesystem may further comprise a fluid flow unit 610. The fluid flow unitmay comprise any element associated with fluid flow described herein,such as any or all of elements 330, 335, 340, 345, 350, 355, 360, 365,430, 435, 440, 445, and 370 described herein with respect to system 300,400, 500 a, or 500 b. The fluid flow unit may be configured to direct asolution comprising a plurality of nucleotides described herein to anarray of the substrate prior to or during rotation of the substrate. Thefluid flow unit may be configured to direct a washing solution describedherein to an array of the substrate prior to or during rotation of thesubstrate. In some instances, the fluid flow unit may comprise pumps,compressors, and/or actuators to direct fluid flow from a first locationto a second location. With respect to method 1400, the fluid flow systemmay be configured to direct any solution to the substrate 310. Withrespect to method 1400, the fluid flow system may be configured tocollect any solution from the substrate 310. The system may furthercomprise a detector 370, such as any detector described herein withrespect to system 300 or 400. The detector may be in sensingcommunication with the array of the substrate.

The system may further comprise one or more computer processors 620. Theone or more processors may be individually or collectively programmed toimplement any of the methods described herein. For instance, the one ormore processors may be individually or collectively programmed toimplement any or all operations of the methods of the presentdisclosure, such as method 200 or 1400. In particular, the one or moreprocessors may be individually or collectively programmed to: (i) directthe fluid flow unit to direct the solution comprising the plurality ofnucleotides across the array during or prior to rotation of thesubstrate; (ii) subject the nucleic acid molecule to a primer extensionreaction under conditions sufficient to incorporate at least onenucleotide from the plurality of nucleotides into a growing strand thatis complementary to the nucleic acid molecule; and (iii) use thedetector to detect a signal indicative of incorporation of the at leastone nucleotide, thereby sequencing the nucleic acid molecule.

While the rotational system has been described with respect tosequencing applications, such rotational schemes may be used for otherapplications (e.g., pre-sequencing applications, sample preparation,etc.), such as template seeding and surface amplification processes. Forexample, the reagents dispensed during or prior to rotation of thesubstrate may be tailored to the other applications. While the reagentsdispensed to the substrate in the rotational system have been describedwith respect to nucleotides, any reagent that may react with a nucleicacid molecule (or any other molecule or cell) immobilized to thesubstrate, such as probes, adaptors, enzymes, and labelling reagents,may be dispensed to the substrate prior to, during, or subsequent torotation to achieve high speed coating of the substrate with thedispensed reagents.

The systems described herein (such as any of systems 300, 400, 500 a, or500 b, or any other system described herein), or any element thereof,may be environmentally controlled. For instance, the systems may bemaintained at a specified temperature or humidity. The systems (or anyelement thereof) may be maintained at a temperature of at least 20degrees Celsius (° C.), at least 25° C., at least 30° C., at least 35°C., at least 40° C., at least 45° C., at least 50° C., at least 55° C.,at least 60° C., at least 65° C., at least 70° C., at least 75° C., atleast 80° C., at least 85° C., at least 90° C., at least 95° C., atleast 100° C., at most 100° C., at most 95° C., at most 90° C., at most85° C., at most 80° C., at most 75° C., at most 70° C., at most 65° C.,at most 60° C., at most 55° C., at most 50° C., at most 45° C., at most40° C., at most 35° C., at most 30° C., at most 25° C., at most 20° C.,or at a temperature that is within a range defined by any two of thepreceding values.

Different elements of the system may be maintained at differenttemperatures or within different temperature ranges, such as thetemperatures or temperature ranges described herein. Elements of thesystem may be set at temperatures above the dew point to preventcondensation. Elements of the system may be set at temperatures belowthe dew point to collect condensation.

FIG. 7 illustrates a system with different environmental conditions inan open substrate system. An open substrate system may comprise asubstrate 3502 and a container 3504 enclosing the substrate. Thesubstrate 3502 may be any substrate described herein. The container 3504may define a surrounding environment of the substrate 3502. In someinstances, the surrounding environment may be confined and/or closed. Insome instances, the surrounding environment may be sealed (e.g.,hermetically sealed, frictionally sealed, pneumatically, etc.). In someinstances, the surrounding environment may be sealed using a pressuredifferential (e.g., pneumatic pressure, mechanical pressure, etc.). Theopen substrate system may comprise at least two non-overlapping regions,a first region 3522 and a second region 3524, having differentenvironmental conditions. In some instances, the first region 3522,contacting or in proximity to a surface of the substrate 3502, such asthe surface that comprises one or more analytes as described herein, maybe maintained at a first set of temperatures and first set ofhumidities. In some instances, the second region 3524, contacting or inproximity to a top portion of the container 3504 (or otherwise referredto herein as a lid or cover), may be maintained at a second set oftemperatures and second set of humidities. The first set of temperaturesand first set of humidities may be controlled such as to prevent orminimize evaporation of one or more reagents on the surface of thesubstrate. For example, the first set of temperatures and first set ofhumidities may be configured to prevent less than 80%, less than 70%,less than 60%, less than 50%, less than 40%, less than 30%, less than20%, less than 10%, less than 5%, less than 4%, less than 3%, less than2%, or less than 1% evaporation of the volume of the solution layerdispensed on the uncovered surface. The second set of temperatures andsecond set of humidities may also be controlled such as to enhance orrestrict condensation. For example, the first set of temperatures may bethe lowest temperatures within the surrounding environment of the opensubstrate system. For example, the second set of temperatures may be thehighest temperatures within the surrounding environment of the opensubstrate system. In some instances, the environmental conditions of thedifferent regions may be achieved by controlling the temperature of theenclosure. In some instances, the environmental conditions of thedifferent regions may be achieved by controlling the temperature ofselected parts or whole of the container. In some instances, theenvironmental conditions of the different regions may be achieved bycontrolling the temperature of selected parts or whole of the substrate.In some instances, the environmental conditions of the different regionsmay be achieved by controlling the temperature of reagents dispensed tothe substrate. Any combination thereof may be used to control theenvironmental conditions of the different regions. Heat transfer may beachieved by any method, including for example, conductive, convective,and radiative methods. For example, the first region 3522 may bemaintained at cooler temperatures by controlling the temperature of thesubstrate 3502, and the second region 3524 may be maintained at warmertemperatures by controlling the temperature of a top portion of thecontainer 3504, via conduction.

The system may further comprise a reservoir beneath the substrate 3522(not shown in FIG. 7). The reservoir may be configured to hold fluid.The reservoir may be configured to collect fluid, precipitation, orcondensation from other surfaces, for example from the substrate 3522 orthe top portion of the container 3504. Fluid may be removed from thereservoir. In some cases, fluid may be removed from the reservoirvolumetrically. For example, fluid may be removed from the reservoirvolumetrically to balance an amount of fluid added to the system. Insome cases, fluid is continuously added to the system and fluid iscontinuously removed from the reservoir. The amount of fluid added maybe equal to the amount of fluid removed. In some cases, a volume offluid in the reservoir is held constant. The volume of fluid in thereservoir may be determined based on a relative humidity of the system.The relative humidity of the system may depend on the volume of fluid inthe reservoir, the amount of fluid in the system, the temperature of thesystem, or any combination thereof.

The system may be temperature controlled. In some cases, the elements ofthe system may be held at different temperatures. The differentialtemperatures of individual elements in the system may control theaccumulation of condensation or precipitation on the individual elementsof the system. The top portion of the container 3504 may be held at adifferent temperature than the substrate 3502, an objective (forexample, as shown in FIG. 15), or the reservoir. Alternatively or inaddition, the substrate may be held at a different temperature than thetop portion of the container, the objective, or the reservoir.Alternatively or in addition, the reservoir may be held at a differenttemperature than the top portion of the container, the objective, or thesubstrate. Alternatively or in addition, the objective may be held at adifferent temperature than the top portion of the container, thereservoir, or the substrate. In some cases, the top portion of thecontainer is held at a higher temperature than at least one otherelement in the system to prevent the accumulation of condensation on thetop surface of the container. In an exemplary configuration, the topportion of the container is held at the highest temperature, thesubstrate is held at the lowest temperature, and the reservoir and theobjective are held at intermediate temperatures, thereby preventingcondensation from forming on the top portion of the container or fromforming or dripping onto the objective. In another example, theobjective is held at the highest temperature, the top portion of thecontainer is held at an intermediate temperature, and the substrate andthe reservoir are held at lower temperatures than the top portion of thecontainer, thereby preventing condensation from forming on the topportion of the container or from forming or dripping onto the objective.In some cases, the objective may be fully or partially surrounded by aseal. The seal may be configured to prevent moisture from the containersurrounding the substrate (for example, as shown in FIG. 7) fromreaching other optical components in the system (for example, asdescribed with respect to FIG. 41). The seal may comprise a flexiblematerial. The flexible seal may be configured to allow relative motionof individual elements of the system while maintaining the seal. In someembodiments, the flexible seal may stretch, expand, or contract. Forexample, the flexible seal may be configured to allow independent motionof two or more imaging heads, as described with respect to FIG. 29F-FIG.29G. Alternatively or in addition, the seal may comprise a waterproofmaterial. For example, the seal may be rubber, silicone, latex, plastic,Teflon, nitrile, elastin, an elastomer, or a polymer. The seal maysurround the objective and contact the top portion of the container. Insome cases, a portion of the objective comprising a front lens is notcovered by the seal. The front lens of the objective may be exposed tothe container surrounding the substrate. In some cases, the front lensof the objective may be in fluidic contact with the substrate.

The systems (or any element thereof) may be maintained at a relativehumidity of at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, at most 100%, at most 95%, at most 90%, at most 85%, at most80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, atmost 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most25%, at most 20%, at most 15%, at most 10%, at most 5%, or a relativehumidity that is within a range defined by any two of the precedingvalues. The systems (or any element thereof) may be configured such thatless than 80%, less than 70%, less than 60%, less than 50%, less than40%, less than 30%, less than 20%, less than 10%, less than 5%, lessthan 4%, less than 3%, less than 2%, or less than 1% of the volume ofthe solution layer dispensed on the uncovered surface evaporates.

The systems (or any element thereof) may be contained within a sealedcontainer, housing, or chamber that insulates the system (or any elementthereof) from the external environment or atmosphere, allowing for thecontrol of the temperature or humidity. An environmental unit (e.g.,humidifiers, heaters, heat exchangers, compressors, etc.) may beconfigured to regulate one or more operating conditions in eachenvironment. In some instances, each environment may be regulated byindependent environmental units. In some instances, a singleenvironmental unit may regulate a plurality of environments. In someinstances, a plurality of environmental units may, individually orcollectively, regulate the different environments. An environmental unitmay use active methods or passive methods to regulate the operatingconditions. For example, the temperature may be controlled using heatingor cooling elements. The humidity may be controlled using humidifiers ordehumidifiers. In some instances, a part of the internal environmentwithin the container or chamber may be further controlled from otherparts of the internal environment. Different parts may have differentlocal temperatures, pressures, and/or humidity. For example, theinternal environment may comprise a first internal environment and asecond internal environment separated by a seal.

Alternatively or in conjunction, the systems or methods described hereinmay comprise a solution comprising an agent that may reduce evaporation.For example, the solution may comprise glycerol, which can preventevaporation of the solution.

In some instances, the seal may comprise an immersion objective lens,which is described in further detail elsewhere herein. For example, animmersion objective lens may be part of a seal that separates theinternal environment in the container into a first internal environmenthaving 100% (or substantially 100%) humidity and a second environmenthaving one or more of an ambient temperature, pressure or humidity. Theimmersion objective lens may be in contact with one or more of adetector and imaging lens.

Substrate Preparation and Contaminant-Resistant Substrates

As described above, a substrate may comprise a surface comprising aplurality of binders coupled thereto. In some cases, the plurality ofbinders may comprise a plurality of nucleic acid molecules (e.g., aplurality of nucleic acid molecules that are directly coupled to thesurface or that are indirectly coupled to the surface via a plurality oflinkers, as described herein). Oligonucleotide (e.g., nucleic acidmolecule)-coated surfaces (e.g., substantially planar substrates and/orparticles, including substrates having a plurality of particlesimmobilized thereto) may be employed for various applications, includingfor capturing specific sequences of nucleic acid molecules for, e.g.,gene expression analysis by hybridization capture (gene arrays), singlenucleotide polymorphism (SNP) genotyping, capturing a subset ofsequencing libraries (e.g., targeted capture or exome sequencing),synthesis of cDNA from mRNA via oligo-dT capture, and on-surfaceamplification of nucleic acid molecules for downstream analysis such asnext generation sequencing. An oligonucleotide-coated surface may beprepared in advance of its use in any such application and may be storedbetween its generation and its eventual use (e.g., during transport froma manufacturing site to an operating site, sample processing andpreparation, etc.). An oligonucleotide-coated surface may be stored forat least 1 hour, and in some cases may be stored for months or evenyears. During storage, an oligonucleotide-coated surface may come intocontact with one or more solutions or other materials that may containnucleic acid molecules, which may be considered contaminants.Contaminant nucleic acid molecules may hybridize to oligonucleotidescoupled to a surface, leading to decreased efficiency in downstreamanalysis (e.g., during use in an application such as those describedherein) and/or erroneous results in downstream analysis. For example, anoligonucleotide-coated surface prepared for use in a sequencing analysismay become contaminated with non-relevant sequencing libraries duringhandling of the surface prior to its use in the sequencing analysis(e.g., prior to placement of the substrate comprising the surface in asequencing instrument or to commencement of an amplification process,such as a clonal amplification process).

Non-relevant interactions of oligonucleotides (e.g., binders) coupled toa surface of a substrate may be reduced by blocking the oligonucleotidesthat are attached to the surface (e.g., bound oligonucleotides) witholigonucleotides comprising sequences that are fully or partiallycomplementary to the sequences of the oligonucleotides that are attachedto the surface. Blocking oligonucleotides may be provided in solutionand may be considered “free” oligonucleotides. For example, blockingoligonucleotides may fully or partially hybridize to all or a subset ofthe oligonucleotides coupled to a surface of a substrate, therebyproviding a partially double-stranded nucleic acid molecule comprising abound oligonucleotide and a blocking oligonucleotide. Such a partiallydouble-stranded nucleic acid molecule may be resistant to hybridizationto nucleic acid molecules with which the surface may come into contact,including potential contaminant nucleic acid molecules that may not berelevant to any eventual analysis such as eventual nucleic acidsequencing. Blocking oligonucleotides may be removed from theoligonucleotide-coated surface (e.g., via application of an appropriatestimulus, such as a chemical or thermal stimulus, or via enzymaticdegradation) to provide an oligonucleotide-coated surface that may beready to use in an analysis process (e.g., as described herein). Thesurface may undergo one or more washing processes (e.g., one or morewash flows) to remove blocking oligonucleotides. Removing the blockingoligonucleotides may provide the oligonucleotides coupled to the surfaceas free oligonucleotides that may participate in various reactions,including capture of complementary or partially complementary nucleicacid molecules of interest.

An oligonucleotide-coated surface may be stored for any useful amount oftime. For example, an oligonucleotide-coated surface may be stored forat least 1 hour, such as at least 2 hours, 6 hours, 12 hours, 24 hours,36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 1 year, or longer. Anoligonucleotide-coated surface may be stored under any usefulconditions. For example, an oligonucleotide-coated surface may be storedunder standard temperature and pressure conditions (e.g., roomtemperature), such as between about 18° C. to about 30° C., such asbetween about 20° C. to about 25° C., and about 1 atmosphere. Anoligonucleotide-coated surface may be stored in a dry environment (e.g.,in air or a nitrogen- or argon-enriched environment) or in a solution(e.g., a buffered solution such as saline sodium citrate).

An oligonucleotide-coated surface may be stored in a package orcontainer, which package or container may contain one or more sucholigonucleotide-coated surfaces. For example, multipleoligonucleotide-coated surfaces may be provided in a given package orcontainer. A package or container comprising one or moreoligonucleotide-coated surfaces may be a rigid package or container or aflexible package or container. For example, one or moreoligonucleotide-coated surfaces, such as one or more substantiallyplanar substrates comprising the one or more oligonucleotide-coatedsurfaces, may be provided in a flexible package. A package or containermay comprise or be formed of, for example, a glass, plastic polymer,metal (e.g., metal foil), or any other material. A package or containercomprising one or more oligonucleotide-coated surfaces may be sealed(e.g., hermetically sealed). A package or container comprising one ormore oligonucleotide-coated surfaces may be resealable upon opening. Forexample, a first oligonucleotide-coated surface may be removed from thepackage or container and a second oligonucleotide-coated surface may beretained within the package or container. An oligonucleotide-coatedsurface may also be configured for storage outside of a package orcontainer for a period of time, such as for at least about 1 hour, 2hours, 6 hours, or longer (e.g., as described herein).

An oligonucleotide-coated surface may be prepared at a manufacturingand/or shipping site. Alternatively, an oligonucleotide-coated surfacemay be prepared by a user, such as a user of a sequencing instrument. Insome cases, an oligonucleotide-coated surface comprising a plurality ofblocking oligonucleotides coupled (e.g., hybridized) to a plurality ofoligonucleotides coupled to the oligonucleotide-coated surface may beprepared at a manufacturing and/or shipping site. Alternatively, anoligonucleotide-coated surface comprising a plurality of blockingoligonucleotides coupled (e.g., hybridized) to a plurality ofoligonucleotides coupled to the oligonucleotide-coated surface may beprepared by a user, such as a user of a sequencing instrument. Aplurality of blocking oligonucleotides coupled to a plurality ofoligonucleotides coupled to the oligonucleotide-coated surface may beremoved by a user, such as a user of a sequencing instrument. Forexample, a plurality of blocking oligonucleotides coupled to a pluralityof oligonucleotides coupled to the oligonucleotide-coated surface may beremoved by a user shortly before a user makes use of theoligonucleotide-coated surface (e.g., as described herein, such as for asequencing application).

An oligonucleotide-coated surface may be used one or more times for oneor more applications. For example, an oligonucleotide-coated surface maybe configured for one-time use. Alternatively, an oligonucleotide-coatedsurface may be configured to be used multiple times, for the same and/ordifferent applications. For example, oligonucleotides coupled to asurface may be “recharged” for use in a subsequent application, or asurface may be washed clean and new oligonucleotides may be coupled tothe surface for use in a subsequent application. In another example, anoligonucleotide-coated surface may comprise one or more differentregions comprising one or more different oligonucleotides (e.g.,binders) coupled thereto (e.g., as described herein). The one or moredifferent oligonucleotides may be configured for use in one or moredifferent applications. In an example, an oligonucleotide-coated surfacecomprises a first plurality of oligonucleotides coupled to a firstregion and a second plurality of oligonucleotides coupled to a secondregion, where the first plurality of oligonucleotides and the secondplurality of oligonucleotides have different nucleic acid sequences. Thefirst plurality of oligonucleotides may be configured to at leastpartially hybridize to a first plurality of blocking oligonucleotides,while the second plurality of oligonucleotides may be configured to atleast partially hybridize to a second plurality of blockingoligonucleotides, where the first plurality of blocking oligonucleotidesand the second plurality of blocking oligonucleotides have differentnucleic acid sequences. The first plurality of blocking oligonucleotideshybridized to the first plurality of oligonucleotides coupled to thesurface may be removable upon application of a first stimulus (e.g., asdescribed herein) and the second plurality of blocking oligonucleotideshybridized to the second plurality of oligonucleotides coupled to thesurface may be removable upon application of a second stimulus, whichsecond stimulus differs from the first stimulus. Accordingly, the firstand second pluralities of blocking oligonucleotides may be provided tothe oligonucleotide-coated surface (e.g., at the same or differenttimes) to provide a doubly-treated surface. The first plurality ofblocking oligonucleotides hybridized to oligonucleotides of the firstplurality of oligonucleotides coupled to the surface may be removed(e.g., after a first period of storage) by application of the firststimulus to provide the first plurality of oligonucleotides coupled tothe first region free to participate in a first application such as afirst sequencing assay. Application of the first stimulus may not affectthe second plurality of blocking oligonucleotides coupled to the secondplurality of oligonucleotides coupled to the second region. Accordingly,the second plurality of blocking oligonucleotides hybridized tooligonucleotides of the second plurality of oligonucleotides coupled tothe surface may be retained during the duration of the firstapplication. The second plurality of blocking oligonucleotideshybridized to oligonucleotides of the second plurality ofoligonucleotides coupled to the surface may be removed (e.g., after asecond period of storage) by application of the second stimulus toprovide the second plurality of oligonucleotides coupled to the secondregion free to participate in a second application such as a secondsequencing assay.

Oligonucleotides may be coupled to an oligonucleotide-coated surface viaany useful mechanism, including, for example, non-specific interactions(e.g., one or more of hydrophilic interactions, hydrophobicinteractions, electrostatic interactions, physical interactions (forinstance, adhesion to pillars or settling within wells), and the like)or specific interactions (e.g., as described herein).

Oligonucleotides may be coupled to an oligonucleotide-coated surfacerandomly or semi-randomly. Alternatively, oligonucleotides may becoupled to an oligonucleotide-coated surface in a predetermined pattern(e.g., as described herein). In some cases, a substrate comprising anoligonucleotide-coated surface may comprise one or more differentbinders (e.g., dispersed with a plurality of oligonucleotides ordisposed on a different region of the substrate). For example, asubstrate comprising an oligonucleotide-coated surface may comprise afirst set of oligonucleotides coupled to the surface and a second set ofoligonucleotides coupled to the surface, where the oligonucleotides ofthe first set of oligonucleotides have a nucleic acid sequence thatdiffers from a nucleic acid sequence of oligonucleotides of the secondset of oligonucleotides. In an example, oligonucleotides of the firstset of oligonucleotides may comprise a first nucleic acid sequence andoligonucleotides of the second set of oligonucleotides may comprise asecond nucleic acid sequence that differs from the first nucleic acidsequence. In some cases, oligonucleotides of the first set ofoligonucleotides and oligonucleotides of the second set ofoligonucleotides may comprise a common third nucleic acid sequence, suchas a poly(T) sequence.

Oligonucleotides may be coupled to one or more particles immobilized toa surface of a substrate. For example, a surface of a substrate maycomprise a plurality of particles (e.g., beads) immobilized thereto(e.g., as described herein), which plurality of particles comprise aplurality of oligonucleotides coupled thereto. In some cases, eachparticle comprises a different plurality of oligonucleotides coupledthereto (e.g., a plurality of oligonucleotides comprising a nucleic acidsequence that differs from a nucleic acid sequence of another pluralityof oligonucleotides coupled to a different particle). For example, eachparticle of a plurality of particles to a surface of a substrate maycomprise a plurality of oligonucleotides coupled thereto, where all ofthe oligonucleotides coupled to a given particle comprise a commonbarcode sequence and where each plurality of oligonucleotides coupled toeach different particle of the plurality of particles comprises adifferent barcode sequence (e.g., as described herein).

An oligonucleotide-coated surface may comprise any useful number ofoligonucleotides coupled thereto (e.g., as described herein). Forexample, an oligonucleotide-coated surface may comprise at least 10,100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000 or moreoligonucleotides. In some cases, an oligonucleotide-coated surfacecomprises multiple regions comprising multiple different pluralities ofoligonucleotides, which different pluralities of oligonucleotides mayhave the same or different nucleic acid sequences and may comprise thesame or different numbers of oligonucleotides. For example, anoligonucleotide-coated surface may comprise a first region comprising afirst plurality of oligonucleotides and a second region comprising asecond plurality of oligonucleotides, where the first plurality ofoligonucleotides and/or the second plurality of oligonucleotidescomprises at least 10, 100, 1000, 10,000, 100,000, 1,000,000,10,000,000, 100,000,000 or more oligonucleotides. The density ofoligonucleotides coupled to a region of a surface may be, for example,at least about 1,000 molecules per mm², such as at least about 10,000molecules per mm², 100,000 molecules per mm², 1,000,000 molecules permm², 10,000,000 molecules per mm², or more. The density ofoligonucleotides coupled to a surface may vary by region. For example, asurface may comprise a first region comprising a first density ofoligonucleotides coupled thereto and a second region comprising a seconddensity of oligonucleotides coupled thereto, where the first density ishigher than the second density.

Oligonucleotides coupled to a surface of a substrate may comprise one ormore different nucleic acid sequences. For example, an oligonucleotidecoupled to a surface of a substrate may comprise a barcode sequence, anadapter sequence, a primer sequence (e.g., a universal primer sequence),a poly(T) sequence, a random N-mer sequence, a flow cell adaptersequence, a sequencing primer, a unique molecular identifier, a keysequence, an index sequence, or any other useful sequence. One or moresequences of an oligonucleotide coupled to a surface may be configuredto capture a particular sample molecule or population thereof. In somecases, an oligonucleotide-coated surface may comprise a plurality ofoligonucleotides coupled thereto, wherein each oligonucleotide of theplurality of oligonucleotides comprises at least one common or sharedsequence. For example, each oligonucleotide of a plurality ofoligonucleotides coupled to an oligonucleotide-coated surface or a givenregion thereof may comprise a common barcode sequence. Alternatively orin addition, each oligonucleotide of the plurality of oligonucleotidescoupled to an oligonucleotide-coated surface or a given region thereofmay comprise a poly(T) sequence (e.g., for capture of sample nucleicacid molecules comprising a poly(A) sequence, such as mRNA molecules) oranother specific capture sequence. In some cases, each oligonucleotideof a plurality of oligonucleotides coupled to an oligonucleotide-coatedsurface or a region thereof may comprise one or more common sequences(e.g., as described herein) and a different unique molecular identifieror key sequence.

Oligonucleotides coupled to a surface of a substrate may have any usefullength. For example, an oligonucleotide coupled to a surface of asubstrate may comprise at least 6 bases, such as 7, 8, 9, 10, 12, 14,16, 18, 20, 25, 30, 35, or more bases. In some cases, only a portion ofthe bases of an oligonucleotide coupled to a surface of a substrate maybe accessible to a blocking or other oligonucleotide. For example, oneor more nucleotides of an oligonucleotide coupled to a surface of asubstrate may comprise a blocking moiety and/or may be coupled to othermoieties, such as a moiety immobilizing the oligonucleotide to thesurface. In some cases, an oligonucleotide coupled to a surface of asubstrate may comprise one or more reversible terminators.

Similarly, a blocking oligonucleotide may have any useful length. Forexample, a blocking oligonucleotide may comprise at least 6 bases, suchas 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, or more bases. In somecases, only a portion of the bases of a blocking oligonucleotide may beavailable to hybridize to an oligonucleotide coupled to anoligonucleotide-coated surface. For example, one of more nucleotides ofa blocking oligonucleotide may comprise a blocking moiety and/or may becoupled to other moieties. In some cases, a blocking oligonucleotide maycomprise one or more groups that may be substantially inert orunreactive (e.g., in a buffered solution). In some cases, a blockingoligonucleotide may comprise one or more reversible terminators.

Oligonucleotides coupled to a surface of a substrate may have any usefulcomposition. Oligonucleotides coupled to a surface may comprisenucleotides, nucleotide analogs, nonstandard nucleotides, and/ormodified analogs (e.g., as described herein). For example,oligonucleotides coupled to a surface may comprise DNA nucleotides, RNAnucleotides, and/or a mixture thereof. Similarly, blockingoligonucleotides coupled to a surface may have any useful composition,provided that the blocking oligonucleotides comprise a nucleic acidsequence that is fully or partially complementary to oligonucleotidescoupled to a surface of a substrate. Blocking oligonucleotides maycomprise DNA nucleotides, RNA nucleotides, and/or a mixture thereof. Inan example, an oligonucleotide coupled to a surface comprises DNAnucleotides and a blocking oligonucleotide configured to hybridizepartially or completely to the oligonucleotide coupled to the surfacecomprises DNA nucleotides. In another example, an oligonucleotidecoupled to a surface comprises RNA nucleotides and a blockingoligonucleotide configured to hybridize partially or completely to theoligonucleotide coupled to the surface comprises RNA nucleotides.

An oligonucleotide coupled to a surface may comprise an adapter orcomplement thereof. For example, an oligonucleotide may comprise asequence complementary to a sequence of an adapter coupled to a samplenucleic acid molecule (e.g., a single-stranded sample nucleic acidmolecule, such as a single-stranded sample RNA molecule). An adapter maybe a single-stranded adapter and may have any useful composition. Forexample, an adapter may comprise DNA nucleotides, RNA nucleotides, or acombination thereof. An adapter may have any useful length and otherproperties. An adapter may be disposed at an end of an oligonucleotidethat is distal from the surface to which the oligonucleotide is coupled.The adapter may comprise a barcode sequence (e.g., as described herein).

An oligonucleotide coupled to a surface and/or a blockingoligonucleotide may comprise a functional feature such as a terminator(e.g., reversible terminator), blocking moiety, or a label or reportermoiety. For example, a blocking oligonucleotide may comprise a labelmoiety such as a fluorescent label (e.g., a dye, as described herein). Alabel moiety or other functional feature may be linked to a nucleotideof an oligonucleotide via a linker moiety. For example, a nucleotide ofa blocking oligonucleotide may comprise a label moiety (e.g., dye)linked to the base of the nucleotide via a linker moiety. The nucleotidemay be disposed at an end of the blocking oligonucleotide. Alternativelyor in addition, a nucleotide of a blocking oligonucleotide may comprisea terminator (e.g., reversible terminator). The terminator may be linkedto the sugar of the nucleotide via a linker moiety. The nucleotide maybe disposed at the end of the blocking oligonucleotide. Such functionalfeatures may facilitate control of the interaction between blockingoligonucleotides and oligonucleotides coupled to a surface of asubstrate and/or provide a mechanism for identifying where blockingoligonucleotides have hybridized to oligonucleotides coupled to asurface. Alternatively or in addition, an oligonucleotide coupled to asurface may comprise a label or reporter moiety, which label or reportermoiety may emit a first signal when the oligonucleotide is uncoupled anda second signal when the oligonucleotide is coupled to a blockingoligonucleotide. For example, the second signal may be attenuated,decreased, quenched, or amplified relative to the first signal. In somecases, no detectable signal may be emitted by the label or reportermoiety when the oligonucleotide coupled to the surface is hybridized toa blocking oligonucleotide. In this manner, coupling betweenoligonucleotides coupled to a surface and blocking oligonucleotides maybe monitored (e.g., to gauge the blocking efficiency of the blockingoligonucleotides). For example, oligonucleotides coupled a surface mayeach comprise a dye that emits a signal when the oligonucleotides are“free,” which signal is severely attenuated when the oligonucleotidesare “blocked” (e.g., hybridized to blocking oligonucleotides). Byoptically interrogating the surface before and after provision of theblocking oligonucleotides, the blocking efficiency of the blockingoligonucleotides (and thus the contamination resistance of the treatedsurface) can be gauged. In some cases, different fluorescent dyes may beused for different areas of a surface (e.g., for oligonucleotides havingdifferent nucleic acid sequences that may be coupled to different areasof the surface).

A treated surface comprising a plurality of oligonucleotides immobilizedthereto and a plurality of blocking oligonucleotides coupled tooligonucleotides of the plurality of oligonucleotides may have anydegree of “contamination resistance.” The percentage of oligonucleotidesof the plurality of oligonucleotides that are coupled to blockingoligonucleotides of the plurality of blocking oligonucleotides may beindicative of the resistance of the treated surface to contamination. Insome cases, at least 50% of the oligonucleotides of the plurality ofoligonucleotides may be coupled to blocking oligonucleotides. Forexample, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or more of the oligonucleotides of the plurality ofoligonucleotides may be coupled to blocking oligonucleotides. Couplingbetween oligonucleotides coupled to the surface and blockingoligonucleotides may be monitored via optical detection (e.g., asdescribed herein) or any other useful method.

FIG. 38A-FIG. 38D illustrate a blocking oligonucleotide scheme. In FIG.38A, a substrate comprising bound oligonucleotides is provided. In FIG.38B, the bound oligonucleotides are blocked using blockingoligonucleotides (e.g., as described herein). As shown in FIG. 38C,contaminant nucleic acid molecules cannot bind to the boundoligonucleotides while the blocking oligonucleotides are coupled to thebound oligonucleotides. FIG. 38D shows removal of the blockingoligonucleotides using various mechanisms, including heat denaturation,chemical denaturation, chemical degradation, and enzymatic degradation.After the blocking oligonucleotides have been removed, relevant targetnucleic acid molecules (e.g., from a sample for various applicationssuch as sequencing) may be able to bind to substrate-boundoligonucleotides (e.g., substrate-bound oligonucleotides comprisingsequences that are at least partially complementary to the targetnucleic acid molecules, as described herein).

In an aspect, the present disclosure provides a method for storing asubstrate comprising a nucleic acid molecule-coated surface. A substratehaving a surface comprising a first set of nucleic acid moleculesimmobilized thereto may be provided. Nucleic acid molecules of the firstset of nucleic acid molecules may be configured to capture samplenucleic acid molecules derived from one or more nucleic acid samples(e.g., nucleic acid samples for sequencing). The substrate comprisingthe surface comprising the first set of nucleic acid molecules may bebrought into contact with a second set of nucleic acid molecules underconditions sufficient to yield a treated surface in which at least 70%(e.g., at least 75%, 80%, 85%, 90%, or more) of nucleic acid moleculesof the first set of nucleic acid molecules may be hybridized to nucleicacid molecules of the second set of nucleic acid molecules, wherein thesecond set of nucleic acid molecules are not the sample nucleic acidmolecules. Excess nucleic acid molecules of the second set of nucleicacid molecules may be washed away. The substrate having the treatedsurface may be stored for a period of time, such as at least 1 hour, 6hours, 12 hours, 24 hours, 2 days, or longer. The treated surface may bestored under any useful conditions (e.g., as described herein). Duringstorage of the treated surface, each nucleic acid molecule of the firstset of nucleic acid molecules that is hybridized to a nucleic acidmolecule of the second set of nucleic acid molecules may not hybridizeto another nucleic acid molecule.

The second set of nucleic acid molecules may be provided to the surfaceof the substrate in a solution. Each nucleic acid molecule of the secondset of nucleic acid molecules may comprise a sequence that issubstantially complementary to a sequence of the first set of nucleicacid molecules. The sequence of the first set of nucleic acid moleculesmay comprise at least 6 bases, such as at least 10 bases, 20 bases, ormore. Each nucleic acid molecule of the first set of nucleic acidmolecules may comprise at least 6 bases, such as at least 10 bases, 20bases, or more. The first set of nucleic acid molecules and/or thesecond set of nucleic acid molecules may comprise DNA nucleotides, RNAnucleotides, or a combination thereof. Each nucleic acid molecule of thefirst set of nucleic acid molecules may comprise the same nucleic acidsequence. In some cases, the first set of nucleic acid molecules maycomprise one or more different nucleic acid sequences. The first set ofnucleic acid molecules may comprise a first subset of nucleic acidmolecules comprising a first nucleic acid sequence and a second subsetof nucleic acid molecules comprising a second nucleic acid sequence,which first and second nucleic acid sequences are different. The firstsubset of nucleic acid molecules and the second subset of nucleic acidmolecules may both comprise a third nucleic acid sequence. The thirdnucleic acid sequence may comprise a poly(T) sequence.

The nucleic acid molecules of the first set of nucleic acid moleculesmay be immobilized to the surface at independently addressablelocations. The independently addressable locations may be substantiallyplanar and may comprise one or more wells. Nucleic acid molecules of thefirst set of nucleic acid molecules may be immobilized to the surface ofthe substrate according to a predetermined pattern. A density of thefirst set of nucleic acid molecules on the surface may be at least10,000 molecules per mm², such as at least 100,000, 1,000,000,10,000,000, or more molecules per mm². The surface of the substrate maybe substantially planar. The substrate may comprise one or moreparticles immobilized thereto.

The method may further comprise, subsequent to a period of storage ofthe treated surface, removing nucleic acid molecules of the second setof nucleic acid molecules from the treated surface. The nucleic acidmolecules may be removed via, for example, enzymatic degradation or viadenaturing via chemical or thermal stimulation (e.g., application of achemical stimulus such as sodium hydroxide). After removing thesenucleic acid molecules, the first set of nucleic acid moleculesimmobilized to the surface may be used for, e.g., hybridization capture,single nucleotide polymorphism (SNP) genotyping, sequencing librarycapture, synthesis of nucleic acid molecules, on-surface amplification,downstream processing or analysis of nucleic acid molecules orderivatives thereof, or combinations thereof.

In another aspect, the present disclosure provides a method forpreparing a substrate having a treated surface for use in nucleic acidprocessing. A substrate having a treated surface may be provided, whichsubstrate comprises a first set of nucleic acid molecules immobilizedthereto. At least 70% (e.g., at least 80%, 85%, 90%, 95%, or more) ofnucleic acid molecules of the first set of nucleic acid molecules may behybridized to nucleic acid molecules of a second set of nucleic acidmolecules. Nucleic acid molecules of the first set of nucleic acidmolecules may be configured to capture sample nucleic acid moleculesderived from one or more nucleic acid samples. The second set of nucleicacid molecules is distinct from the sample nucleic acid molecules. Thesubstrate having the treated substrate may have been stored for a timeperiod of at least 1 hour, such as at least 6 hours, 12 hours, 24 hours,2 days, or longer. The treated surface may have been stored under anyuseful conditions (e.g., as described herein). During storage of thetreated surface, each nucleic acid molecule of the first set of nucleicacid molecules that is hybridized to a nucleic acid molecule of thesecond set of nucleic acid molecules may not hybridize to anothernucleic acid molecule.

Nucleic acid molecules of the second set of nucleic acid molecules fromthe treated surface may be removed (e.g., as described herein). Forexample, the nucleic acid molecules may be removed from the treatedsurface via enzymatic degradation or via denaturing via chemical orthermal stimulation (e.g., application of a chemical stimulus such assodium hydroxide). After removing these nucleic acid molecules, thefirst set of nucleic acid molecules immobilized to the surface may beused for, e.g., hybridization capture, single nucleotide polymorphism(SNP) genotyping, sequencing library capture, synthesis of nucleic acidmolecules, on-surface amplification, downstream processing or analysisof nucleic acid molecules or derivatives thereof, or combinationsthereof.

Each nucleic acid molecule of the second set of nucleic acid moleculesmay comprise a sequence that is substantially complementary to asequence of the first set of nucleic acid molecules. The sequence of thefirst set of nucleic acid molecules may comprise at least 6 bases, suchas at least 10 bases, 20 bases, or more. Each nucleic acid molecule ofthe first set of nucleic acid molecules may comprise at least 6 bases,such as at least 10 bases, 20 bases, or more. The first set of nucleicacid molecules and/or the second set of nucleic acid molecules maycomprise DNA nucleotides, RNA nucleotides, or a combination thereof.Each nucleic acid molecule of the first set of nucleic acid moleculesmay comprise the same nucleic acid sequence. In some cases, the firstset of nucleic acid molecules may comprise one or more different nucleicacid sequences. The first set of nucleic acid molecules may comprise afirst subset of nucleic acid molecules comprising a first nucleic acidsequence and a second subset of nucleic acid molecules comprising asecond nucleic acid sequence, which first and second nucleic acidsequences are different. The first subset of nucleic acid molecules andthe second subset of nucleic acid molecules may both comprise a thirdnucleic acid sequence. The third nucleic acid sequence may comprise apoly(T) sequence.

The nucleic acid molecules of the first set of nucleic acid moleculesmay be immobilized to the surface at independently addressablelocations. The independently addressable locations may be substantiallyplanar and may comprise one or more wells. Nucleic acid molecules of thefirst set of nucleic acid molecules may be immobilized to the surface ofthe substrate according to a predetermined pattern. A density of thefirst set of nucleic acid molecules on the surface may be at least10,000 molecules per mm², such as at least 100,000, 1,000,000,10,000,000, or more molecules per mm². The surface of the substrate maybe substantially planar. The substrate may comprise one or moreparticles immobilized thereto.

In another aspect, the present disclosure provides a method for storinga substrate comprising a nucleic acid molecule-coated surface,comprising providing a substrate having a surface comprising a first setof nucleic acid molecules immobilized thereto. Nucleic acid molecules ofthe first set of nucleic acid molecules may be configured to capturesample nucleic acid molecules derived from one or more nucleic acidsamples. Each nucleic acid molecule of the nucleic acid molecules of thefirst set of nucleic acid molecules may comprise a first nucleic acidsequence. A second set of nucleic acid molecules may also be provided,wherein each nucleic acid molecule of the second set of nucleic acidmolecules comprises a second nucleic acid sequence that may besubstantially complementary to the first nucleic acid sequence. Thesecond set of nucleic acid molecules may be distinct from the samplenucleic acid molecules. The surface comprising the first set of nucleicacid molecules may be brought into contact with the second set ofnucleic acid molecules to generate a treated surface in which at least70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or more) of nucleic acidmolecules of the first set of nucleic acid molecules may be hybridizedto nucleic acid molecules of the second set of nucleic acid molecules.For each nucleic acid molecule of the first set of nucleic acidmolecules hybridized to a nucleic acid molecule of the second set ofnucleic acid molecules, the first nucleic acid sequence may behybridized to the second nucleic acid sequence. The first nucleic acidsequence hybridized to the second nucleic acid sequence may at leastpartially denature between about 40° C. and 60° C., such as betweenabout 50° C. and 60° C. The treated surface may then be stored for aperiod of time, such as for at least one hour, 2 hours, 6 hours, 12hours, 24 hours, 2 days, or longer. The treated surface may be storedunder any useful conditions (e.g., as described herein). During storageof the treated surface, each nucleic acid molecule of the first set ofnucleic acid molecules that is hybridized to a nucleic acid molecule ofthe second set of nucleic acid molecules may not hybridize to anothernucleic acid molecule.

The second set of nucleic acid molecules may be provided to the surfaceof the substrate in a solution. The first nucleic acid sequence and thesecond nucleic acid sequence may each comprise at least 6 bases, such asat least 10 bases, 20 bases, or more. Each nucleic acid molecule of thesecond set of nucleic acid molecules may comprise at least 6 bases, suchas at least 10 bases, 20 bases, or more. Similarly, each nucleic acidmolecule of the first set of nucleic acid molecules may comprise atleast 6 bases, such as at least 10 bases, 20 bases, or more. A givennucleic acid molecule of the first set of nucleic acid molecules and agiven nucleic acid molecule of the second set of nucleic acid moleculesmay comprise the same number of nucleotides. Alternatively, a givennucleic acid molecule of the first set of nucleic acid molecules and agiven nucleic acid molecule of the second set of nucleic acid moleculesmay comprise a different number of nucleotides. The first set of nucleicacid molecules and/or the second set of nucleic acid molecules maycomprise DNA nucleotides, RNA nucleotides, or a combination thereof. Insome cases, the first set of nucleic acid molecules may comprise one ormore different nucleic acid sequences. The first set of nucleic acidmolecules may comprise a first subset of nucleic acid moleculescomprising the first nucleic acid sequence and a second subset ofnucleic acid molecules comprising a third nucleic acid sequence, whichfirst and third nucleic acid sequences are different. The first subsetof nucleic acid molecules and the second subset of nucleic acidmolecules may both comprise a fourth nucleic acid sequence. The fourthnucleic acid sequence may comprise a poly(T) sequence.

The nucleic acid molecules of the first set of nucleic acid moleculesmay be immobilized to the surface at independently addressablelocations. The independently addressable locations may be substantiallyplanar and may comprise one or more wells. Nucleic acid molecules of thefirst set of nucleic acid molecules may be immobilized to the surface ofthe substrate according to a predetermined pattern. A density of thefirst set of nucleic acid molecules on the surface may be at least10,000 molecules per mm², such as at least 100,000, 1,000,000,10,000,000, or more molecules per mm². The surface of the substrate maybe substantially planar and may comprise a plurality of wells. Thesubstrate may comprise one or more particles immobilized thereto.

The method may further comprise, subsequent to a period of storage ofthe treated surface, removing nucleic acid molecules of the second setof nucleic acid molecules from the treated surface. The nucleic acidmolecules may be removed via, for example, enzymatic degradation or viadenaturing via chemical or thermal stimulation (e.g., application of achemical stimulus such as sodium hydroxide). The nucleic acid moleculesof the second set of nucleic acid molecules may be removed from saidtreated surface by denaturing said first nucleic acid sequencehybridized to the second nucleic acid sequence, e.g., by heating thetreated surface or a solution in contact with the treated surface tobetween about 40° C. and 60° C. After removing these nucleic acidmolecules, the first set of nucleic acid molecules immobilized to thesurface may be used for, e.g., hybridization capture, single nucleotidepolymorphism (SNP) genotyping, sequencing library capture, synthesis ofnucleic acid molecules, on-surface amplification, downstream processingor analysis of nucleic acid molecules or derivatives thereof, orcombinations thereof.

In some cases, a single nucleic acid molecule may play the role of botha nucleic acid molecule coupled to a surface and a blocking nucleic acidmolecule. For example, a nucleic acid molecule coupled to a surface maycomprise a first sequence and a second sequence, which second sequencemay be complementary to the first sequence. The second sequence mayhybridize to the first sequence to provide a hairpin molecule that isimmobilized to the surface. Such a scheme may provide a higher blockingefficiency and thus a higher contamination resistance. The portion ofthe nucleic acid molecule including the second sequence may be separatedfrom the immobilized portion of the nucleic acid molecule including thefirst sequence (e.g., by cleaving the molecule at a cleavage sitedisposed between the first and second sequences) and the portion of thenucleic acid molecule including the second sequence may be removed(e.g., via denaturation or enzymatic degradation) and washed away.

Accordingly, in another aspect, the present disclosure may provide amethod for storing a substrate comprising a nucleic acid molecule-coatedsurface, comprising providing a substrate having a surface comprising afirst set of nucleic acid molecules immobilized thereto, wherein nucleicacid molecules of the first set of nucleic acid molecules may beconfigured to capture sample nucleic acid molecules derived from one ormore nucleic acid samples. Each nucleic acid molecule of the first setof nucleic acid molecules may comprise a first nucleic acid sequence anda second nucleic acid sequence, which second nucleic acid sequence issubstantially complementary to the first nucleic acid sequence. Thefirst sequence and the second sequence may each comprise at least 6bases, such as at least 10 bases, 12 bases, 15 bases, 20 bases, or more.A treated surface may be generated by subjecting the surface toconditions sufficient to bind the first nucleic acid sequence of anucleic acid molecule of the first set of nucleic acid molecules to thesecond nucleic acid sequence of the nucleic acid molecule to provide animmobilized hairpin molecule. The substrate having the treated surfacemay then be stored for a time period of at least 1 hour, such as atleast 2 hours, 6 hours, 12 hours, 24 hours, 2 days, or longer. Thetreated surface may be stored under any useful conditions (e.g., asdescribed herein). During storage of the treated surface, each nucleicacid molecule of the first set of nucleic acid molecules may nothybridize to another nucleic acid molecule. At least 70% (e.g., at least75%, 80%, 85%, 90%, 95%, or more) of nucleic acid molecules of the firstset of nucleic acid molecules may be present as immobilized hairpinmolecules during storage of the treated surface.

The nucleic acid molecules of the first set of nucleic acid moleculesmay be immobilized to the surface at independently addressablelocations. The independently addressable locations may be substantiallyplanar and may comprise one or more wells. Nucleic acid molecules of thefirst set of nucleic acid molecules may be immobilized to the surface ofthe substrate according to a predetermined pattern. A density of thefirst set of nucleic acid molecules on the surface may be at least10,000 molecules per mm², such as at least 100,000, 1,000,000,10,000,000, or more molecules per mm². The surface of the substrate maybe substantially planar, and/or may comprise a plurality of wells. Thesubstrate may comprise one or more particles immobilized thereto.

The first set of nucleic acid molecules may comprise one or moredifferent nucleic acid sequences. For example, the first set of nucleicacid molecules may comprise a first subset of nucleic acid moleculescomprising the first nucleic acid sequence and the second nucleic acidsequence, and a second subset of nucleic acid molecules comprising athird nucleic acid sequence and a fourth nucleic acid sequence. Thethird nucleic acid sequence may be substantially complementary to thefourth nucleic acid sequences. The first nucleic acid sequence may bedifferent from the third and fourth nucleic acid sequences. The firstsubset of nucleic acid molecules and the second subset of nucleic acidmolecules may both comprise a fifth nucleic acid sequence, which fifthnucleic acid sequence may comprise a poly(T) sequence.

The method may further comprise, subsequent to storage of the treatedsurface for a period of time, separating the second sequence from thefirst sequence of the immobilized hairpin molecule. Separating the firstand second sequences may be achieved via enzymatic degradation ordenaturation using a chemical or thermal stimulus (e.g., a chemicalstimulus such as sodium hydroxide). After separating these sequences,the first set of nucleic acid molecules immobilized to the surface maybe used for, e.g., hybridization capture, single nucleotide polymorphism(SNP) genotyping, sequencing library capture, synthesis of nucleic acidmolecules, on-surface amplification, downstream processing or analysisof nucleic acid molecules or derivatives thereof, or combinationsthereof. Each nucleic acid molecule of the first set of nucleic acidmolecules may comprise a cleavable base. The cleavable base may bedisposed between the first and second sequences of the nucleic acidmolecule. Subsequent to separating the first and second sequences of theimmobilized hairpin molecule, the nucleic acid molecule may be cleavedat the cleavable base, thereby removing the second sequence of thenucleic acid molecule from the surface.

The present disclosure also provides kits including treated surfaces andkits for preparing treated surfaces. A kit may include a substratecomprising a treated surface and one or more reagents for processing thetreated surface (e.g., for removing blocking oligonucleotides from thetreated surface and preparing the surface for use in a subsequentapplication). A kit may include a substrate comprising a surface and aplurality of oligonucleotides for coupling to the substrate. The kit mayalso include a plurality of blocking oligonucleotides configured tohybridize to the plurality of oligonucleotides, as well as reagents forremoving the blocking oligonucleotides and/or preparing the surface foruse in a subsequent application. A kit provided herein may also comprisereagents for use in a subsequent application, and/or instructions forstoring, preparing, unblocking, or otherwise utilizing a surface of asubstrate.

In an aspect, the present disclosure provides a kit comprising asubstrate comprising a treated surface, wherein the treated surfacecomprises a plurality of pairs of bound nucleic acid molecules, whereineach pair of the plurality of pairs comprises a first nucleic acidmolecule of a first set of nucleic acid molecules at least partiallyhybridized to a second nucleic acid molecule of a second set of nucleicacid molecules. The first set of nucleic acid molecules may beimmobilized to the surface. At least 70% (e.g., 75%, 80%, 85%, 90%, 95%,or more) of nucleic acid molecules of the first set of nucleic acidmolecules may be paired with a nucleic acid molecule of the second setof nucleic acid molecules. Nucleic acid molecules of the first set ofnucleic acid molecules may be configured to capture sample nucleic acidmolecules derived from one or more nucleic acid samples when the nucleicacid molecules of the first set of nucleic acid molecules are not pairedwith nucleic acid molecules of the second set of nucleic acid molecules.

The treated surface may be stored for a period of time, such as for atleast 6 hours, 12 hours, 24 hours, 2 days, or longer. The treatedsurface may be stored under any useful conditions (e.g., as describedherein). During storage of the treated surface, each nucleic acidmolecule of the first set of nucleic acid molecules in each pair of theplurality of pairs may not hybridize to another nucleic acid molecule(e.g., a sample nucleic acid molecule).

The kit may comprise one or more reagents for processing nucleic acidmolecules. For example, the kit may comprise a kit further comprising achemical stimulus (e.g., sodium hydroxide) configured to remove secondnucleic acid molecules from the treated surface.

The surface of the substrate may be substantially planar, and/or maycomprise a plurality of wells. In some cases, the substrate may compriseone or more particles (e.g., beads) immobilized thereto. Nucleic acidmolecules of the first set of nucleic acid molecules may be immobilizedto the surface at independently addressable locations. The independentlyaddressable locations may be substantially planar, and/or may compriseone or more wells. In some cases, a density of the first set of nucleicacid molecules on the surface may be at least 10,000 molecules per mm²,such as at least 100,000, 1,000,000, 10,000,000, or more molecules permm². Nucleic acid molecules of the first set of nucleic acid moleculesmay be immobilized to the surface according to a predetermined patternor may be randomly distributed on the surface.

The second nucleic acid molecule may comprise a sequence that issubstantially complementary to a sequence of the first nucleic acidmolecule. The sequence of the first nucleic acid molecule and/or thesecond nucleic acid molecule may comprise at least 6 bases, such as atleast 10, 12, 16, 20, or more bases. In some cases, the first nucleicacid molecule and the second nucleic acid molecule may comprise the samenumber of nucleotides. Alternatively, the first nucleic acid moleculeand the second nucleic acid molecule may comprise different numbers ofnucleotides. Each nucleic acid molecule of the second set of nucleicacid molecules may comprise at least 6 bases. The first and/or secondset of nucleic acid molecules may comprise DNA nucleotides, RNAnucleotides, or a mixture thereof.

Each nucleic acid molecule of the first set of nucleic acid moleculesmay comprise the same nucleic acid sequence. Alternatively, the firstset of nucleic acid molecules may comprise one or more different nucleicacid sequences. For example, the first set of nucleic acid molecules maycomprise a first subset of nucleic acid molecules comprising a firstnucleic acid sequence and a second subset of nucleic acid moleculescomprising a second nucleic acid sequence. The first and second nucleicacid sequences may be different. The first and second subsets of nucleicacid molecules may both comprise a third nucleic acid sequence, whichthird nucleic acid sequence may comprise a poly(T) sequence.

In another aspect, the present disclosure provides a kit comprising asubstrate comprising a surface comprising a first set of nucleic acidmolecules immobilized thereto, wherein the first set of nucleic acidmolecules comprises one or more first nucleic acid molecules. One ormore first nucleic acid molecules may be configured to capture samplenucleic acid molecules derived from one or more nucleic acid samples.The kit may also comprise a solution comprising a second set of nucleicacid molecules, wherein the second set of nucleic acid moleculescomprises one or more second nucleic acid molecules, which one or moresecond nucleic acid molecules are not said sample nucleic acidmolecules. The second set of nucleic acid molecules may be selected suchthat, upon bringing the solution in contact with the surface, at least70% of the one or more first nucleic acid molecules (e.g., at least 75%,80%, 85%, 90%, 90%, or more) bind to a second nucleic acid molecule ofthe second set of nucleic acid molecules to generate one or more pairsof bound nucleic acid molecules. Each pair of the one or more pairs maycomprise (i) a first nucleic acid molecule of the first set of nucleicacid molecules and a second nucleic acid molecule of the second set ofnucleic acid molecules, and (ii) a section of substantiallycomplementary sequences. Each nucleic acid molecule of the first set ofnucleic acid molecules in each pair of the one or more pairs may nothybridize to another nucleic acid molecule (e.g., during storage of thetreated surface). For example, paired nucleic acid molecules may nothybridize to a sample nucleic acid molecule.

The treated surface may be stored for a period of time, such as for atleast 6 hours, 12 hours, 24 hours, 2 days, or longer. The treatedsurface may be stored under any useful conditions (e.g., as describedherein).

The kit may comprise one or more reagents for processing nucleic acidmolecules. For example, the kit may comprise a kit further comprising achemical stimulus (e.g., sodium hydroxide) configured to remove secondnucleic acid molecules from the treated surface.

The surface of the substrate may be substantially planar, and/or maycomprise a plurality of wells. In some cases, the substrate may compriseone or more particles (e.g., beads) immobilized thereto. Nucleic acidmolecules of the first set of nucleic acid molecules may be immobilizedto the surface at independently addressable locations. The independentlyaddressable locations may be substantially planar, and/or may compriseone or more wells. In some cases, a density of the first set of nucleicacid molecules on the surface may be at least 10,000 molecules per mm²,such as at least 100,000, 1,000,000, 10,000,000, or more molecules permm². Nucleic acid molecules of the first set of nucleic acid moleculesmay be immobilized to the surface according to a predetermined patternor may be randomly distributed on the surface.

The section of substantially complementary sequences of each pair of theone or more pairs may comprise a first sequence of a first nucleic acidmolecule of the one or more first nucleic acid molecules and a secondsequence of a second nucleic acid molecule of the one or more secondnucleic acid molecules. The first sequence may be substantiallycomplementary to the second sequence. The first and second sequences mayeach comprise the same number of bases. In some cases, the first andsecond sequences may each comprise between about 6-20 bases. A firstnucleic acid molecule of the one or more first nucleic acid moleculesand a second nucleic acid molecule of the one or more second nucleicacid molecules may comprise the same number of nucleotides.Alternatively, a first nucleic acid molecule of the one or more firstnucleic acid molecules and a second nucleic acid molecule of the one ormore second nucleic acid molecules may comprise different numbers ofnucleotides. Each nucleic acid molecule of the second set of nucleicacid molecules may comprise at least 6 bases. The first and/or secondset of nucleic acid molecules may comprise DNA nucleotides, RNAnucleotides, or a mixture thereof. The sequence of a nucleic acidmolecule of the first nucleic acid molecules and/or a nucleic acidmolecule of the second nucleic acid molecules may comprise at least 6bases, such as at least 10, 12, 16, 20, or more bases.

Each nucleic acid molecule of the first set of nucleic acid moleculesmay comprise the same nucleic acid sequence. Alternatively, the firstset of nucleic acid molecules may comprise one or more different nucleicacid sequences. For example, the first set of nucleic acid molecules maycomprise a first subset of nucleic acid molecules comprising a firstnucleic acid sequence and a second subset of nucleic acid moleculescomprising a second nucleic acid sequence. The first and second nucleicacid sequences may be different. The first and second subsets of nucleicacid molecules may both comprise a third nucleic acid sequence, whichthird nucleic acid sequence may comprise a poly(T) sequence.

Optical Systems for Imaging a Rotating Substrate

For a substrate exhibiting a smooth, stable rotational motion, it may besimpler or more cost-effective to image the substrate using a rotationalmotion system instead of a rectilinear motion system. Rotational motion,as used herein, may generally refer to motion in a polar coordinatesystem, comprising an angular component φ, and a radial component r,that is predominantly in an angular direction, φ. Prior optical imagingsystems have utilized time delay and integration (TDI) cameras toachieve high duty cycles and maximum integration times per field point.A TDI camera (e.g., a TDI line-scan camera) may use a detectionprinciple similar to a charge coupled device (CCD) camera. Compared to aCCD camera, the TDI camera may shift electric charge, row by row, acrossa sensor at the same rate as an image traverses the focal plane of thecamera. In this manner, the TDI camera may allow longer imageintegration times while reducing artifacts such as blurring that may beotherwise associated with long image exposure times. A TDI camera mayperform integration while simultaneously reading out and may thereforehave a higher duty cycle than a camera that performs these functions ina serial manner. Use of a TDI camera to extend integration times may beimportant for high throughput fluorescent samples, which may be limitedin signal production by fluorescent lifetimes. For instance, alternativeimaging techniques, such as point scanning, may be precluded from use inhigh throughput systems as it may not be possible to acquire an adequatenumber of photons from a point in the limited amount of integration timerequired for high speeds due to limits imposed by fluorescence lifetimesof dye molecules.

FIG. 8A-FIG. 8D illustrate example schemes for a line-scan camera. Asshown in FIG. 8A, a TDI line-scan camera may comprise two or morevertically arranged rows of pixels (such as 3, 4, 5, 6, 7, 8, 9, 10, 24,36, 48, 50, 60, 72, 84, 96, 100, 108, 120, 128, 132, 150, 200, 256, 512,1024, 2000, 2048, 4000, 4096, 8000, 8192, 12000, 16000, 16384, or morepixels). During operation of the camera (e.g., movement of the camerarelative to an open substrate), photoelectrons from each pixel in agiven row may be summed into the row below the given row (e.g., in thedirection of relative object motion) by shifting accumulated chargesbetween pixel rows. FIG. 8B and FIG. 8C show pixel schemes for use incolor line-scan cameras. Such cameras may include rows of pixels havingdifferent color filters to detect and/or block light of differentwavelengths. For example, FIG. 8B shows a trilinear pixel schemeincluding rows of red, green, and blue filters. This trilinear pixelscheme may be replicated one or more times to facilitate TDIapplications. FIG. 8C shows a bilinear pixel scheme including a row ofalternating red and blue filters and a row of green filters. FIG. 8Dshows an alternative bilinear pixel scheme including multiple Bayerpatterns (e.g., 2×2 pixel arrays including a first row alternating blueand green pixels and a second row alternating green and red pixels).Like the trilinear scheme, the bilinear patterns may be replicated oneor more times to facilitate TDI applications. The color line-scanschemes depicted in FIG. 8B-FIG. 8D may be substituted by alternativecolor combinations, including cyan, yellow, green, and magenta; red,green, blue, and emerald; cyan, magenta, yellow, and white; or any othercolor combination, in any arrangements (e.g., alternating,non-alternating).

Prior TDI detection schemes may be limited in their applicability to theimaging of rotating systems, such as the rotating nucleic acidsequencing systems described herein. When scanning a curved path, suchas the curved path generated by the rotating systems described herein, aTDI sensor may only be able to shift charge (commonly referred to asclocking or line triggering) at the correct rate for a single velocity.For instance, the TDI sensor may only be able to clock at the correctrate along an arc located at a particular distance from the center ofrotation. Locations at smaller distances from the center of rotation mayclock too quickly, while locations at smaller distances from the centerof rotation may clock too slowly. In either case, the mismatch betweenthe rotational speed of the rotating system and the clock rate of theTDI sensor may cause blurring that varies with the distance of alocation from the center of the rotating system. This effect may bereferred to as tangential velocity blur. The tangential velocity blurmay produce an image distortion of a magnitude a defined by equation(2):

$\begin{matrix}{\sigma = {\frac{hw}{2R} = \frac{A}{2R}}} & (2)\end{matrix}$

Here, h, w, and A are the effective height, width, and area,respectively, of the TDI sensor projected to the object plan. Thesevalues may be adjusted using one of more optical elements (e.g., lenses,prisms, mirrors, etc.). R is the distance of the center of the fieldfrom the center of the rotating system. The effective height, width, andarea of the sensor are the height, width, and area, respectively, thatproduce signal. In the case of fluorescence imaging, the effectiveheight, width, and area of the sensor may be the height, width, andarea, respectively, that correspond to illuminated areas on the sample.In addition to the tangential velocity blur effect, Equation (2) impliesthat increasing sensor area, which may be a goal of many imagingsystems, may introduce imaging complications for TDI imaging of rotatingsystems. Consequently, prior TDI systems may require small image sensorsto image rotating systems and may thus be unfit for simultaneoushigh-sensitivity and high-throughput imaging of such systems.

Described herein are systems and methods for imaging rotating systemsthat can address at least the abovementioned problems. The systems andmethods described herein may benefit from higher efficiency, such asfrom faster imaging time.

FIG. 9 shows an optical system 700 for continuous area scanning of asubstrate during rotational motion of the substrate. The term“continuous area scanning (CAS),” as used herein, generally refers to amethod in which an object in relative motion is imaged by repeatedly,electronically or computationally, advancing (clocking or triggering) anarray sensor at a velocity that compensates for object motion in thedetection plane (focal plane). CAS can produce images having a scandimension larger than the field of the optical system. TDI scanning maybe an example of CAS in which the clocking entails shiftingphotoelectric charge on an area sensor during signal integration. For aTDI sensor, at each clocking step, charge may be shifted by one row,with the last row being read out and digitized. Other modalities mayaccomplish similar function by high speed area imaging and co-additionof digital data to synthesize a continuous or stepwise continuous scan.

The optical system may comprise one or more sensors 710. As shown, inFIG. 9, the sensors may detect an image optically projected from thesample. The optical system may comprise one or more optical elements,such as the optical element 810 described in the context of FIG. 8. Anoptical element may be, for example, a lens, prism, mirror, wave plate,filter, attenuator, grating, diaphragm, beam splitter, diffuser,polarizer, depolarizer, retroreflector, spatial light modulator, or anyother optical element. The system may comprise a plurality of sensors,such as at least 2, at least 5, at least 10, at least 20, at least 50,at least 100, at least 200, at least 500, or at least 1,000 sensors. Thesystem may comprise a at least 2, at least 4, at least 8, at least 16,at least 32, at least 64, at least 128, at least 256, at least 512, orat least 1,024 sensors. The plurality of sensors may be the same type ofsensor or different types of sensors. Alternatively, the system maycomprise at most about 1000, 500, 200, 100, 50, 20, 10, 5, 2, or fewersensors. Alternatively, the system may comprise at most about 1024, 512,256, 128, 64, 32, 16, 8, 4, 2, or fewer sensors. The system may comprisea number of sensors that is within a range defined by any two of thepreceding values. The sensors may comprise image sensors. The sensorsmay comprise CCD cameras. The sensors may comprise CMOS cameras. Thesensors may comprise TDI cameras (e.g., TDI line-scan cameras). Thesensors may comprise pseudo-TDI rapid frame rate sensors. The sensorsmay comprise CMOS TDI or hybrid cameras. The sensors may be integratedtogether in a single package. The sensors may be integrated together ina single semiconductor substrate. The system may further comprise anyoptical source described herein (not show in FIG. 9).

The sensors may be configured to detect an image from a substrate, suchas the substrate 310 described herein, during rotational motion of thesubstrate. The rotational motion may be with respect to an axis of thesubstrate. The axis may be an axis through the center of the substrate.The axis may be an off-center axis. The substrate may be configured torotate at any rotational speed described herein. The rotational motionmay comprise compound motion. The compound motion may comprise rotationand an additional component of radial motion. The compound motion may bea spiral (or substantially spiral). The compound motion may be a ring(or substantially ring-like).

Each sensor may be located at a conjugate focal plane with respect tothe substrate. Each sensor may be in optical communication with thesubstrate. The conjugate focal plane may be the approximate plane in animaging system (e.g., CAS sensor) at which an image of a region of thesubstrate forms. A sensor may be located at a plane conjugate to a planecomprising the substrate (e.g., an image plane). The conjugate focalplane may be segmented into a plurality of regions, such as at least 2,at least 5, at least 10, at least 20, at least 50, at least 100, atleast 200, at least 500, or at least 1000 regions. The conjugate focalplane may be segmented into at least 2, at least 4, at least 8, at least16, at least 32, at least 64, at least 128, at least 256, at least 512,or at least 1,024 regions. The conjugate focal plane may be segmentedinto a number of regions that is within a range defined by any two ofthe preceding values. The conjugate focal plane may be segmented into aplurality of regions along an axis substantially normal to a projecteddirection of the rotational motion. An angle between the axis and theprojected direction of the rotational motion may be no more than 1degree, no more than 2 degrees, no more than 3 degrees, no more than 4degrees, no more than 5 degrees, no more than 6 degrees, no more than 7degrees, no more than 8 degrees, no more than 9 degrees, no more than 10degrees, no more than 11 degrees, no more than 12 degrees, no more than13 degrees, no more than 14 degrees, or no more than 15 degrees fromnormal, or an angle that is within a range defined by any two of thepreceding values. The conjugate focal plane may be segmented into aplurality of regions along an axis parallel to a projected direction ofthe rotational motion. The conjugate focal plane may be spatiallysegmented. For instance, the conjugate focal plane may be segmented byabutting or otherwise arranging a plurality of sensors in a single focalplane and clocking each of the sensors independently.

Alternatively or in combination, the conjugate focal plane may besegmented by optically splitting the conjugate focal plane into aplurality of separate focal paths, each of which may form a sub-image onan independent sensor of the plurality of sensors and which may beclocked independently. The focal path may be optically split using oneor more optical elements, such as a lens array, mirror, or prism. Eachsensor of the plurality of sensors may be in optical communication witha different region of the rotating substrate. For instance, each sensormay image a different region of the rotating substrate. Each sensor ofthe plurality of sensors may be clocked at a rate appropriate for theregion of the rotating substrate imaged by the sensor, which may bebased on the distance of the region from the center of the rotatingsubstrate or the tangential velocity of the region.

One or more of the sensors may be configured to be in opticalcommunication with at least 2 of the plurality of regions in theconjugate focal plane. One or more of the sensors may comprise aplurality of segments. Each segment of the plurality of segments may bein optical communication with a region of the plurality of regions. Eachsegment of the plurality of segments may be independently clocked. Theindependent clocking of a segment may be linked to a velocity of animage in an associated region of the focal plane. The independentclocking may comprise TDI line rate or pseudo-TDI frame rate.

The system may further comprise a controller (not shown). The controllermay be operatively coupled to the one or more sensors. The controllermay be programmed to process optical signals from each region of therotating substrate. For instance, the controller may be programmed toprocess optical signals from each region with independent clockingduring the rotational motion. The independent clocking may be based atleast in part on a distance of each region from a projection of the axisand/or a tangential velocity of the rotational motion. The independentclocking may be based at least in part on the angular velocity of therotational motion. While a single controller has been described, aplurality of controllers may be configured to, individually orcollectively, perform the operations described herein.

FIG. 10A shows an optical system 800 for imaging a substrate duringrotational motion of the substrate using tailored optical distortions.The optical system may comprise one or more sensors 710. The one or moresensors may comprise any sensors described herein. The optical systemmay comprise any optical sources described herein (not shown in FIG.10A). FIG. 10B shows optical system 801 for imaging a substrate duringrotational motion of the substrate using tailored optical distortions.The optical system may comprise one or more sensors 710. The one or moresensors may comprise any sensors described herein. The optical systemmay comprise any optical sources described herein (not shown in FIG.10B). The optical system may comprise a lens 810, for example aplano-convex lens. In some embodiments, the substrate 310 is tilted withrespect to the lens 810 and the detector 710. In some embodiments, thelens 810 is tilted with respect to the detector 710, thereby producinganamorphic magnification of light (e.g., fluorescence or scatteredlight) from the substrate. Anamorphic magnification may result indifferential magnification of light from a first region of the substrate820 and a second region of the substrate 830. The light from the firstregion of the substrate may be magnified by a first amount at a firstposition on the detector 825, and the light from the second region ofthe substrate may be magnified by a second amount at a second positionon the detector 835. In some embodiments, the anamorphic magnificationmay occur along a single axis. In some embodiments, a cylindrical lensmay be used to produce anamorphic magnification along a single axis.

The sensors may be configured to detect an image from a substrate, suchas the substrate 310 described herein, during rotational motion of thesubstrate. The rotational motion may be with respect to an axis of thesubstrate. The axis may be an axis through the center of the substrate.The axis may be an off-center axis. The substrate may be configured torotate at any rotational speed described herein.

The system 800 may further comprise an optical element 810. The opticalelement may be in optical communication with the sensor. The opticalelement may be configured to direct optical signals from the substrateto the sensor. The optical element may produce an optical magnificationgradient across the sensor. At least one of the optical elements and thesensor may be adjustable. For instance, at least one of the opticalelements and the sensor may be adjustable to generate an opticalmagnification gradient across the sensor. The optical magnificationgradient may be along a direction substantially perpendicular to aprojected direction of the rotational motion of the substrate. Theoptical element may be configured to rotate, tilt, or otherwise bepositioned to engineer the optical magnification gradient. The opticalelement may produce a magnification that scales approximately as theinverse of the distance to the axis of the substrate. The magnificationgradient may be produced by selecting a relative orientation of thesubstrate, optical element, and sensor. For instance, the magnificationgradient may be produced by tilting the object and image planes as shownin FIG. 10A and FIG. 10B. The magnification gradient may displaygeometric properties. For instance, a ratio of a first opticalmagnification of a first region 820 at a maximum distance from thecenter of the substrate to a second optical magnification of a secondregion 830 at a minimum distance from the center of the substrate may besubstantially equal to a ratio of the maximum distance to the minimumdistance. In this manner, the first and second optical magnificationsmay be in the same ratio as the radii of their respective sampleregions. Although the system 800 and system 801 as shown include asingle optical element 810, the system 800 or system 801 may include aplurality of optical elements, such as at least 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 100, or more optical elements. Various arrangementsor configurations of optical elements may be employed. For example, thesystem 800 may include a lens and a mirror for directing light.

The optical element may be a lens. The lens may be a field lens. Thelens may be a cylindrical lens (for instance, as shown in FIG. 10C). Thecylindrical lens may be plano-cylindrical. The lens may be plano-concaveor plano-convex. The cylindrical lens may have a positive or negativecurvature. The curvature of the cylindrical lens may vary. The curvatureof the cylindrical lens may vary in a direction perpendicular to aprojected direction of rotational motion. The shape of a surface of thelens may be conical. The lens may be tilted with respect to the sensor,thereby producing an anamorphic magnification gradient. The tilt of thelens may be adjustable, thereby producing an adjustable anamorphicmagnification gradient.

FIG. 10C shows an example of induced tailored optical distortions usinga cylindrical lens. As shown in FIG. 10C, a cylindrical lens may have afirst side A and a second side B. The first side A may be located closerto an image sensor (such as a TDI camera sensor described herein) thanthe second side B. Such a configuration may be achieved by tilting thecylindrical lens in relation to the image sensor. In this manner, thecylindrical lens may direct light to different locations on the imagesensor, with light passing through side B being directed moredivergently than light passing through side A. In this manner, thecylindrical lens may provide an anamorphic magnification gradient acrossthe image sensor, as depicted in FIG. 10C.

Tilting of the lens may provide an anamorphic magnification gradientacross the sensor. The tilt and hence anamorphic gradient may be in adirection substantially perpendicular to the image motion on the sensor.The tilt of the lens may be adjustable. The adjustment may be automaticby using a controller. The adjustment may be coupled to the radius ofthe scanned substrate region relative to the substrate axis of rotation.The ratio of the minimum to maximum anamorphic magnification may beexactly or approximately in the ratio of the minimum to maximumprojected radii relative to the substrate axis of rotation.

Alternatively or in combination, a gradient in the radius of curvatureof the lens may provide an anamorphic magnification gradient across thesensor. The curvature gradient may be in a direction substantiallydirection perpendicular to the image motion on the sensor.

The system may further comprise a controller (not shown). The controllermay be operatively coupled to the sensor and the optical element. Thecontroller may be programmed to direct the adjustment of at least one ofthe sensors and the optical element to generate an optical magnificationgradient across the sensor. The magnification gradient may be generatedalong a direction substantially perpendicular to a projected directionof the rotational motion. The controller may be programmed to directadjustment of the sensor and/or the optical element to produce ananamorphic optical magnification gradient. The optical magnificationgradient may be across the sensor in a direction substantiallyperpendicular to a projected direction of the rotational motion. Thecontroller may be programmed to direct rotation or tilt of the opticalelement. The controller may be programmed to direct adjustment of themagnification gradient. For instance, the controller may be programmedto direct adjustment of the magnification gradient at least in part on aradial range of a field dimension relative to a projection about theaxis of the substrate. The controller may be programmed to subject therotational motion to the substrate. While a single controller has beendescribed, a plurality of controllers may be configured to, individuallyor collectively, perform the operations described herein.

The optical systems described herein may utilize multiple scan heads.The multiple scan heads may be operated in parallel along differentimaging paths. For instance, the scan heads may be operated to produceinterleaved spiral scans, nested spiral scans, interleaved ring scans,nested ring scans, or a combination thereof. A scan head may compriseone or more of a detector element such as a camera (e.g., a TDIline-scan camera), an illumination source (e.g., as described herein),and one or more optical elements (e.g., as described herein).

FIG. 13A shows a first example of an interleaved spiral imaging scan. Afirst region of a scan head may be operated along a first spiral path910 a. A second region of a scan head may be operated along a secondspiral path 920 a. A third region of a scan head may be operated along athird spiral path 930 a. Each of the first, second, and third regionsmay be independently clocked. The scan head may comprise any opticalsystems described herein. The use of multiple imaging scan paths mayincrease imaging throughput by increasing imaging rate.

FIG. 13B shows a second example of an interleaved spiral imaging scan. Afirst scan head may be operated along a first spiral path 910 b. Asecond scan head may be operated along a second spiral path 920 b. Athird scan head may be operated along a third spiral path 930 b. Each ofthe first, second, and third scan heads may be independently clocked orclocked in unison. Each of the first, second, and third scan heads maycomprise any optical systems described herein. The use of multipleimaging scan paths may increase imaging throughput by increasing netimaging rate. Throughput of the optical system can be multiplied byoperating many scan heads of a field width in parallel. For example,each scan head may be fixed at a different angle relative to the centerof substrate rotation.

FIG. 13C shows an example of a nested spiral imaging scan. A first scanhead may be operated along a first spiral path 910 c. A second scan headmay be operated along a second spiral path 920 c. A third scan head maybe operated along a third spiral path 930 c. Each of the first, second,and third scan heads may be independently clocked. Each of the first,second, and third scan heads may comprise any optical systems describedherein. The use of multiple imaging scan paths may increase imagingthroughput by increasing imaging rate. The scan heads may move togetherin the radial direction. Throughput of the optical system can bemultiplied by operating many scan heads of a field width in parallel.For example, each scan head may be fixed at a different angle. The scansmay be in discrete rings rather than spirals.

While FIG. 13A-FIG. 13C illustrate three imaging paths, there may be anynumber of imaging paths and any number of scan heads. For example, theremay be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more imaging pathsor scan heads. Alternatively, there may be at most about 10, 9, 8, 7, 6,5, 4, 3, 2, or fewer imaging paths or scan heads. Each scan head may beconfigured to receive light having a wavelength within a givenwavelength range. For instance, the first scan head may be configured toreceive first light having a wavelength within a first wavelength range.The second scan head may be configured to receive second light having awavelength within a second wavelength range. The third scan head may beconfigured to receive third light having a wavelength within a thirdwavelength range. Similarly, fourth, fifth, sixth, seventh, eighth,ninth, or tenth scan heads may be configured to receive fourth, fifth,sixth, seventh, eighth, ninth, or tenth light, respectively, each of thefourth, fifth, sixth, seventh, eighth, ninth, or tenth light having awavelength within a fourth, fifth, sixth, seventh, eighth, ninth, ortenth wavelength range, respectively. The first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, or tenth wavelength ranges may beidentical. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth wavelength ranges may partially overlap. Any 2,3, 4, 5, 6, 7, 8, 9, or 10 of the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth wavelength ranges may bedistinct. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth wavelength ranges may be in the ultraviolet,visible, or near infrared regions of the electromagnetic spectrum. Eachof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth wavelength ranges may comprise a wavelength emitted by afluorophore, dye, or quantum dot described herein. In this manner, thesystem may be configured to detect optical signals from a plurality offluorophores, dyes, or quantum dots.

Scanning a surface may comprise detecting a focus of the surfacerelative to the detector. In some embodiments, scanning the surfacecomprises adjusting the focus of the surface relative to the detector.The optical systems of this disclosure may further comprise one or moreautofocus systems to detect the position of the surface relative to anobjective, as described elsewhere herein. An autofocus system maycomprise an autofocus illumination source. The autofocus system maydetect when the surface moves out of focus relative to the detector. Theautofocus system may be configured to send a signal to a focusing systemto adjust a position of the surface relative to the objective, therebyreturning the surface to a focused position relative to the detector. Insome embodiments the autofocus system may map part or all of a surfaceprior to scanning the surface to generate an autofocus map of thesurface. The autofocus map of the surface may comprise surface textures,irregularities, or tilts that may impact the focus. The autofocus map ofthe surface may be used to anticipate a focal position of the surfaceand adjust the position of the surface relative to the objective tocorrect for the surface textures, irregularities, or tilts. In someembodiments, the autofocus system may map a first part of the surface(e.g., a first ring) before scanning the first part of the surface. Themap of the first part of the surface may be used to anticipate andadjust the focus of the surface while scanning the first part of thesurface. The map of the first part of the surface may be used to predictthe focus of the surface while scanning a second part of the surface(e.g., a second ring). The second portion of the surface may be close tothe first part of the surface so that the map of the first part of thesurface may approximate a map of the second part of the surface. Theautofocus system may map the second portion of the surface whilescanning the second portion of the surface. The map of the second partof the surface may be used to anticipate and adjust the focus of thesurface while scanning the third part of the surface (e.g., a thirdring). In some embodiments, a map generated while scanning a third,fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,or more portion of a surface may be used to anticipate and adjust thefocus of the surface while scanning a fourth, fifth, sixth, seventh,eighth, ninth, tenth, eleventh, twelfth, thirtieth, or more part of thesurface, respectively. Sequential surface portions may be positionedclose together such that the map of a preceding part of the surface mayapproximate a map of the following part of the surface. In someembodiments, the autofocus system may map the entire surface beforescanning. In some embodiments, the autofocus system adjust the focuswhile scanning without generating a map.

FIG. 14 shows a nested circular imaging scan. A first scan head 1005 maybe operated along a first approximately circular path 1010. A secondscan head 1015 may be operated along a second approximately circularpath 1020. A third scan head 1025 may be operated along a thirdapproximately circular path 1030. A fourth scan head 1035 may beoperated along a fourth approximately circular path 1040. A fifth scanhead 1045 may be operated along a fifth approximately circular path1050. A sixth scan head 1055 may be operated along a sixth approximatelycircular path 1060. Each of the first, second, third, fourth, fifth, andsixth scan heads may be independently clocked. Each of the first,second, third, fourth, fifth, and sixth scan heads may comprise anyoptical systems described herein. Each of the first, second, third,fourth, fifth, and sixth scan heads may be configured to remain in afixed location during scanning of a substrate. Alternatively, one ormore of the first, second, third, fourth, fifth, and sixth scan headsmay be configured to move during scanning of a substrate. The use of aplurality of scan heads imaging along approximately circular imagingpaths may greatly increase imaging throughput. For instance, theconfiguration of scan heads depicted in FIG. 14 may allow alladdressable locations on a substrate to be imaged during a singlerotation of the substrate. Such a configuration may have the additionaladvantage of simplifying the mechanical complexity of an imaging systemby requiring only one scanning motion (e.g., the rotation of thesubstrate).

While FIG. 14 illustrates six imaging paths and six scan heads, theremay be any number of imaging paths and any number of scan heads. Forexample, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreimaging paths or scan heads. Alternatively, there may be at most about10, 9, 8, 7, 6, 5, 4, 3, 2, or less imaging paths or scan heads. Eachscan head may be configured to receive light having a wavelength withina given wavelength range. For instance, the first scan head may beconfigured to receive first light having a wavelength within a firstwavelength range. The second scan head may be configured to receivesecond light having a wavelength within a second wavelength range. Thethird scan head may be configured to receive third light having awavelength within a third wavelength range. The fourth scan head may beconfigured to receive fourth light having a wavelength within a fourthwavelength range. The fifth scan head may be configured to receive fifthlight having a wavelength within a fifth wavelength range. The sixthscan head may be configured to receive sixth light having a wavelengthwithin a sixth wavelength range. Similarly, seventh, eighth, ninth, ortenth scan heads may be configured to receive seventh, eighth, ninth, ortenth light, respectively, each of the seventh, eighth, ninth, or tenthlight having a wavelength within a seventh, eighth, ninth, or tenthwavelength range, respectively. The first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth wavelength ranges may beidentical. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth wavelength ranges may partially overlap. Any 2,3, 4, 5, 6, 7, 8, 9, or 10 of the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth wavelength ranges may bedistinct. The first, second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth wavelength ranges may be in the ultraviolet,visible, or near infrared regions of the electromagnetic spectrum. Eachof the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth wavelength ranges may comprise a wavelength emitted by afluorophore, dye, or quantum dot described herein. In this manner, thesystem may be configured to detect optical signals from a plurality offluorophores, dyes, or quantum dots.

FIG. 29A-FIG. 29D, FIG. 30A-FIG. 30D, and FIG. 31A-FIG. 31B showadditional examples of imaging schemes involving multiple imaging heads.For example, FIG. 31B shows rotating scan directions of multiple imagingheads due to non-radial motion of a substrate.

FIG. 15 shows a cross-sectional view of an immersion optical system1100. The system 1100 may be used to optically image the substratesdescribed herein. The system 1100 may be integrated with any otheroptical system or system for nucleic acid sequencing described herein(such as any of systems 300, 400, 500 a, 500 b, 700, or 800), or anyelement thereof. The system may comprise an optical imaging objective1110. The optical imaging objective may be an immersion optical imagingobjective. The optical imaging objective may be configured to be inoptical communication with a substrate, such as substrate 310 describedherein. The optical imaging objective may be configured to be in opticalcommunication with any other optical elements described herein. Theoptical imaging objective may be partially or completely surrounded byan enclosure 1120. The enclosure may partially or completely surround asample-facing end of the optical imaging objective. The enclosure andfluid may comprise an interface between the atmosphere in contact withthe substrate and the ambient atmosphere. The atmosphere in contact withthe substrate and the ambient atmosphere may differ in relativehumidity, temperature, and/or pressure. The enclosure may have agenerally cup-like shape or form. The enclosure may be any container.The enclosure may be configured to contain a fluid or immersion fluid1140 (such as water or an aqueous or organic solution) in which theoptical imaging objective is to be immersed. The enclosure may beconfigured to maintain a minimal distance 1150 between the substrate andthe enclosure in order to avoid contact between the enclosure and thesubstrate during rotation of the substrate. In some instances, air or apressure differential may be used to maintain the minimal distance. Theminimal distance may be at least 100 nm, at least 200 nm, at least 500nm, at least 1 μm, at least 2 μm, at least 5 μm, at least 10 μm, atleast 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least500 μm, at least 1 mm, or a distance that is within a range defined byany two of the preceding values. Even with a minimal distance, theenclosure may contain the fluid due to surface tension effects. Thesystem may comprise a fluid flow tube 1130 configured to deliver fluidto the inside of the enclosure. The fluid flow tube may be connected tothe enclosure through an adaptor 1135. The adaptor may comprise athreaded adaptor, a compression adaptor, or any other adaptor. Anelectrical field application unit (not shown) can be configured toregulate a hydrophobicity of one or more surfaces of a container toretain at least a portion of the fluid contacting the immersionobjective lens and the open substrate, such as by applying an electricalfield.

As used herein, the fluid contacting the immersion objective lens may bereferred to as “immersion fluid” or “fluid”. The immersion fluid maycomprise any suitable immersion medium for imaging. For example, theimmersion medium may comprise an aqueous solution. Non-limiting examplesof aqueous immersion fluids include water. In some cases, the aqueoussolution may comprise salts, surfactants, oils and/or any otherchemicals or reagents useful in imaging. In some cases, the immersionmedium comprises an organic solution. Non-limiting examples of organicimmersion fluids include oils, perfluorinated polyethers,perfluorocarbons, and hydrofluorocarbons. In some cases, the immersionfluid may be substantially the same as the wash buffer, as describedelsewhere herein, or any buffer used in the processes described herein.The immersion fluid may be tuned based on the optical requirements ofthe systems and methods described herein. For example, where a highnumerical aperture (NA) is required, the appropriate immersion fluid(e.g., oil) may be used for imaging. In some cases, the immersion fluidmay be selected to match an index of refraction of a solution on thesubstrate (e.g., a buffer), a surface (e.g., a coverslip or thesubstrate), or an optical component (e.g., an objective lens).

The optical imaging objective may be in fluidic contact with an opensubstrate. The open substrate may comprise a layer of fluid covering thesurface of the substrate. The optical imaging objective may beconfigured to scan the surface comprising the layer of fluid. The layerof fluid on the surface may comprise the same fluid as the immersionfluid. The layer of fluid on the surface may comprise a different fluidthan the immersion fluid. The layer of fluid on the surface may bemiscible with the immersion fluid, or the layer of fluid on the surfacemay be immiscible with the immersion fluid. In some cases, the layer offluid is deeper where it contacts the optical imaging objective than atother points on the surface. A portion of the layer of fluid may adhereto the optical imaging objective. In some cases, the portion of thelayer of fluid may move with the optical imaging objective relative tothe substrate during scanning. The optical imaging objective may remainin fluidic contact with the substrate during scanning. The opticalimaging objective may be configured to have a long travel distance in avertical direction relative to the substrate. In some cases, the opticalimaging objective may be configured to lift away from the substrate suchthat the optical imaging objective is no longer in fluidic contact withthe substrate. For example, the optical imaging objective may be liftedaway from the substrate while fluid is being dispensed on the substrate.A portion of the layer of fluid, the immersion fluid, or both may adhereto the optical imaging objective when it leaves fluidic contact with thesubstrate. The portion of the layer of fluid adhering to the opticalimaging objective may prevent bubbles from forming or accumulatingbetween the substrate and the optical imaging objective when the opticalimaging objective re-enters fluidic contact with the substrate.

The optical imaging objective may be configured to scan a side of thesubstrate that does not comprise a layer of fluid. For example, theoptical imaging objective may be configured to scan a bottom surface ofthe substrate. In some cases, the optical imaging objective may not bein fluidic contact with the substrate. For example, the optical imagingobjective may be an air objective.

The fluid may be in contact with the substrate. The optical imagingobjective and enclosure may be configured to provide a physical barrierbetween a first location in which chemical processing operations areperformed and a second location in which detection operations areperformed. In this manner, the chemical processing operations and thedetection operations may be performed with independent operationconditions and contamination of the detector may be avoided. The firstand second locations may have different humidities, temperatures,pressures, or atmospheric admixtures.

A system of the present disclosure may be contained in a container orother closed environment. For example, a container may isolate aninternal environment 1160 from an external environment 1170. Theinternal environment 1160 may be controlled such as to localizetemperature, pressure, and/or humidity, as described elsewhere herein.In some instances, the external environment 1170 may be controlled. Insome instances, the internal environment 1160 may be furtherpartitioned, such as via, or with aid of, the enclosure 1120 toseparately control parts of the internal environment (e.g., firstinternal environment for chemical processing operations, second internalenvironment for detection operations, etc.). The different parts of theinternal environment may be isolated via a seal. For example, the sealmay comprise the immersion objective described herein.

A system of the present disclosure may be configured to analyze adynamic (e.g., rotating or otherwise moving) open substrate (e.g., asdescribed herein) using a stationary detector system. Alternatively oradditionally, one or more components of a detector system may be inmotion. For example, a detector system may comprise a sensor (e.g.,camera) and an illumination source. The sensor may be in motion while anoptical element (e.g., prism) remains stationary. The illuminationsource may move in tandem with the sensor. For example, the sensor maybe a line-scan camera (e.g., a TDI line-scan camera) and theillumination source may be an LED line light or a laser (e.g., a laserhaving a beam expanded to a line), and the illumination source mayilluminate the area being detected by the sensor. The sensor (and,optionally, the illumination source) may rotate at a same or differentrate as the open substrate. In some cases, the sensor (and, optionally,the illumination source) may translate across the open substrate in apredefined pattern, such as a spiral pattern. Alternatively, the sensor(and, optionally, the illumination source) may translate radially acrossthe open substrate. In some cases, the sensor (and, optionally, theillumination source) may remain in a same physical location but mayrotate about a central axis of the detector system or component(s)thereof. In other cases, the illumination source may illuminate an areaof the open substrate that may be larger than an area that is detectableby the sensor in a given scan or collection of scans. However,illumination over a broad swath of the open substrate may promotebleaching of beads and/or fluorophores that may be disposed on the opensubstrate. Accordingly, the illumination source may be configured toilluminate only a limited area of the open substrate at a given time(e.g., an area that may be, at least partially overlaps with, or iswithin an area detectable by the sensor).

In another example, a detector system may comprise a sensor (e.g.,camera), an illumination source, and one or more optical elements (e.g.,lenses, mirrors, prisms, etc.), and the sensor and illumination sourcemay remain stationary while an optical element (e.g., prism) is inmotion. For instance, the optical element may rotate at a same rate asthe open substrate, or the optical element may translate across the opensubstrate (e.g., radially or in a predefined pattern, such as a spiralpattern). In some cases, the optical element may remain in a samephysical location but may rotate about a central axis (e.g., of theoptical element or the detector system). Motion of an optical element ofa detector system relative to an open substrate in motion may have theeffect of enabling detection at one or more different areas of the opensubstrate. For example, the movement of one or more optical elements ofthe detector system may result in illumination of different areas of theopen substrate to permit detection of signal associated with thedifferent areas of the open substrate. Distortions of the illumination(e.g., laser light) and variation in detection sensitivities overdifferent areas of the open substrate may be compensated for viasubsequent processing (e.g., using a processor, as described herein).

Alternatively, a system of the present disclosure may be configured toanalyze a stationary open substrate using a detector system comprisingone or more dynamic components. For example, a detector system maycomprise a sensor (e.g., camera) and an illumination source. The sensormay be in motion while an optical element (e.g., prism) remainsstationary. The illumination source may move in tandem with the sensor.For example, the sensor may be a line-scan camera (e.g., a TDI line-scancamera) and the illumination source may be an LED line light or a laser(e.g., a laser having a beam expanded to a line), and the illuminationsource may illuminate the area being detected by the sensor. The sensor(and, optionally, the illumination source) may rotate (e.g., about acentral axis of the open substrate). In some cases, the sensor (and,optionally, the illumination source) may translate across the opensubstrate in a predefined pattern, such as a spiral pattern.Alternatively, the sensor (and, optionally, the illumination source) maytranslate radially across the open substrate. In some cases, the sensor(and, optionally, the illumination source) may remain in a same physicallocation but may rotate about a central axis of the detector system orcomponent(s) thereof.

In another example, a detector system may comprise a sensor (e.g.,camera), an illumination source, and one or more optical elements (e.g.,lenses, mirrors, prisms, etc.), and the sensor and illumination sourcemay remain stationary while an optical element (e.g., prism) is inmotion. For instance, the optical element may rotate (e.g., about acentral axis of the open substrate or about a central axis of theoptical element or the detector system) or translate across the opensubstrate (e.g., radially or in a predefined pattern, such as a spiralpattern). Motion of an optical element of a detector system relative toa stationary open substrate may have the effect of enabling detection atone or more different areas of the open substrate. For example, themovement of one or more optical elements of the detector system mayresult in illumination of different areas of the open substrate topermit detection of signal associated with the different areas of theopen substrate. Distortions of the illumination (e.g., laser light) andvariation in detection sensitivities over different areas of the opensubstrate may be compensated for via subsequent processing (e.g., usinga processor, as described herein).

A system may be calibrated (e.g., using an open substrate that does notcomprise an analyte, or comprises a known analyte or collection thereof)to facilitate any detection schemes provided herein.

In any of the preceding examples, multiple sensors and/or illuminationsources may be used (e.g., to detect different areas of the opensubstrate, as described herein). The multiple sensors and/orillumination sources may all remain stationary or may all be in motionduring a detection process. In other cases, certain sensors and/orillumination sources may be in motion and other sensors and/orillumination sources may be stationary during a detection process. Someor all sensors may analyze the substrate. For example, only sensors inmotion, or only sensors that are stationary, may detect signals from theopen substrate.

The scan direction of one or more detector systems (e.g., imaging head)may rotate due to non-radial motion of the detector system relative to asubstrate. For example, a detector system may have different tangentialvelocity vectors relative to the substrate while tracing differentimaging paths at different radial positions along the substrate, whichtangential velocity vectors may point in substantially differentdirections. Such an effect may be manifested as a rotation of theimaging field as a first detector system traces a first set of imagingpaths or as a second detector system traces a second set of imagingpaths (see, e.g., FIG. 31A and FIG. 31B).

The present disclosure provides an apparatus in which processing of ananalyte on an open substrate and detection of a signal associated withthe analyte are performed in the same environment. For example, the opensubstrate may be retained in the same or approximately the same physicallocation during processing of an analyte and subsequent detection of asignal associated with a processed analyte. For a system in which thedetector system or a component thereof is in motion during detection,the apparatus may comprise a mechanical component configured to affectmotion of the detector system of component thereof.

The present disclosure also provides an apparatus in which processing ofan analyte on an open substrate and detection of a signal associatedwith the analyte are performed in different environments. For example,the open substrate may be retained in a first physical location duringprocessing of an analyte and the in a second physical location duringdetection of a signal associated with a processed analyte. The opensubstrate may be transferred between various physical locations via, forexample, a mechanical component. In some cases, the open substrate maybe transferred between various physical locations using a robotic arm,elevator mechanism, or another mechanism. The first physical locationmay be disposed, for example, above, below, adjacent to, or across fromthe second physical location. For example, the first physical locationmay be disposed above the second physical location, and the opensubstrate may be transferred between these locations between analyteprocessing and detection. In another example, the first physicallocation may be disposed adjacent to the second physical location, andthe open substrate may be transferred between these locations betweenanalyte processing and detection. The first and second physicallocations may be separated by a barrier, such as a retractable barrier.

FIG. 12A-FIG. 12C illustrate various detection schemes. FIG. 12Aillustrates a scheme involving a system 3900 in which open substrate3910 rotates and detector system 3920 remains stationary duringdetection. Detector system 3920 may comprise line-scan camera (e.g., TDIline-scan camera) 3930 and illumination source 3940. FIG. 12Billustrates an alternative scheme involving a system 3900 in which opensubstrate 3910 remains stationary and detector system 3920 rotatesduring detection. FIG. 12C illustrates a scheme involving an apparatuscomprising a first system 3950 in which open substrate 3910 is subjectedto analyte processing. As shown in FIG. 3, first system 3950 maycomprise a plurality of fluid channels 3960, 3970, 3980, and 3990, whichplurality of fluid channels may comprise a plurality of fluid outletports 3965, 3975, 3985, and 3995. The apparatus may be configured totransfer open substrate 3910 to second system 3900, in which opensubstrate 3910 is configured to remain stationary and detector system3920 is configured to rotate during detection. While examples describedherein provide relative rotational motion of the substrates and/ordetector systems, the substrates and/or detector systems mayalternatively or additionally undergo relative non-rotational motion,such as relative linear motion, relative non-linear motion (e.g.,curved, arcuate, angled, etc.), and any other types of relative motion.

In an aspect, the present disclosure provides a method for analytedetection or analysis comprising providing an open substrate comprisinga central axis (e.g., as described herein). The open substrate may be,for example, a wafer or disc, such as a wafer or disc having one or moresubstances patterning its surface. The open substrate may besubstantially planar. The open substrate may have an array ofimmobilized analytes thereon (e.g., as described herein). Theimmobilized analytes may be immobilized to the array via one or morebinders. The array may comprise at least 100,000 such binders. In somecases, an immobilized analyte of the immobilized analytes may be coupledto a bead, and the bead may be immobilized to the array. An immobilizedanalyte may comprise a nucleic acid molecule.

A solution having a plurality of probes may be delivered (e.g., asdescribed herein) to a region proximal to the central axis to introducethe solution to the open substrate. The solution may be dispersed acrossthe open substrate such that at least one probe of the plurality ofprobes may bind to at least one immobilized analyte of the immobilizedanalytes to form a bound probe. The plurality of probes may comprise aplurality of oligonucleotide molecules. Alternatively, the plurality ofprobes may comprise a plurality of nucleotides or nucleotide analogs.All or a subset of the plurality of nucleotides or nucleotide analogsmay be fluorescently labeled. In an example, the immobilized analytesmay comprise nucleic acid molecules and the plurality of probes maycomprise fluorescently labeled nucleotides, such that at least onefluorescently labeled nucleotide of the fluorescently labelednucleotides binds to at least one nucleic acid molecule of the nucleicacid molecules via nucleotide complementarity binding. All or a subsetof the plurality of nucleotides or nucleotide analogs may comprise thesame base (e.g., the same canonical nucleobase, such as A, T, C, or G).Similarly, all or a subset of the plurality of nucleotides or nucleotideanalogs may be reversibly terminated. Reversible terminators and, insome cases, fluorescent moieties such as dyes, may be cleaved fromnucleotides (e.g., subsequent to their incorporation into a growingnucleic acid strand) using a cleaving agent, which cleaving agent may beincluded in another solution provided to the open substrate (e.g., asdescribed herein). The open substrate may also be provided with a washsolution to remove excess probes and other reagents, which wash solutionmay be dispersed across the open substrate (e.g., during rotation of theopen substrate using at least centrifugal force, as described herein).

After generation of the bound probe, a detector system may be used todetect at least one signal from the bound probe. The detector system maycomprise a line-scan camera (e.g., a TDI line-scan camera) and anillumination source (e.g., an LED line light or a laser, such as acontinuous wave laser). In some cases, the illumination source maycomprise a laser and the detector system may comprise an optical element(e.g., a cylindrical lens) configured to change a shape of a beam (e.g.,Gaussian beam) emitted by the laser (e.g., as described herein). Theopen substrate may comprise a first area and a second area, where thefirst area and the second area comprise subsets of the array ofimmobilized analytes, are at different radial positions of the opensubstrate with respect to the central axis and are spatially resolvableby the detector system. The bound probe may be disposed in the firstarea of the open substrate. The detector system may perform a non-linearscan of the open substrate. The illumination source and the detectorsystem are described in greater detail with respect to FIG. 41.

During the dispersal and delivery processes, the open substrate may berotating (e.g., in a first physical location). The detector system(e.g., sensor and illumination source) may be stationary during theseprocesses.

During the detection process, the open substrate may be stationary. Thesensor and/or the illumination source of the detector system may be inmotion during detection. For example, the sensor and the illuminationsource may be rotating during detection, optionally at the same rate.The sensor and/or the illumination source may rotate about a centralaxis of the open substrate. Alternatively, the sensor and/or theillumination source may rotate about a central axis of the detectorsystem or a component thereof and remain in a same physical location.The sensor and/or illumination source may translate relative to the opensubstrate in a predetermined pattern, such as a spiral pattern.Alternatively, the line-scan camera and/or illumination source maytranslate (e.g., radially translate) across the open substrate. Thedetector system may further comprise a prism (e.g., a Dove prism), whichprism may rotate during the detection process (e.g., about a centralaxis of the open substrate or about a central axis of the detectorsystem or a component thereof while remaining in a same physicallocation). In an example, the prism may rotate or otherwise translaterelative to the open substrate while the sensor and illumination sourceremain stationary. Such a prism may be used to disperse light to andfrom the open substrate, e.g., to disperse light from the illuminationsource to the open substrate and to detect optical signal from the opensubstrate, such as fluorescence.

The detector system may be configured to illuminate an area of the opensubstrate using the illumination source and subsequently detect a signalfrom the area using a sensor (e.g., line-scan camera). For example, theillumination source may illuminate an area of the open substrate (e.g.,a stationary open substrate) prior to its detection by the sensor. Insuch a situation, the sensor and illumination source may move in tandemrelative to the open substrate. One or more optical elements, such asone or more lenses, mirrors, filters, or other optical elements, maymove in tandem with these other components of the detector system (e.g.,to manipulate light provided to or detected from the open substrate).

During the dispersal and/or delivery processes, an additional probe maybe formed, which additional bound probe may be disposed in the secondarea of the open substrate. During detection, at least one signal may bedetected from the additional bound probe at the same time as the atleast one signal from the bound probe. These signals may be detectedwith different sensitivities.

The detector system may compensate for velocity differences at differentradial positions of the array with respect to the central axis within ascanned area. The detector system may comprise an optical imaging systemhaving an anamorphic magnification gradient substantially transverse toa scanning direction along the open substrate, where the anamorphicmagnification gradient may at least partially compensate for tangentialvelocity differences that are substantially perpendicular to thescanning direction. Detection may comprise reading two or more regionson the open substrate at two or more different scan rates, respectively,to at least partially compensate for tangential velocity differences inthe two or more regions. Detection may further comprise using animmersion objective lens in optical communication with the detectorsystem and the open substrate to detect the at least one signal (e.g.,as described herein). The immersion objective lens may be in contactwith a fluid that is in contact with the open substrate. The fluid maybe in a container, and an electric field may be used to regulate ahydrophobicity of one or more surfaces of the container to retain atleast a portion of the fluid contacting the immersion objective lens andthe open substrate.

The delivery and/or dispersal processes may be performed in a firstenvironment having a first operating condition, the detection processmay be performed in a second environment having a second operatingcondition different from the first operating condition. The first andsecond environments may be disposed in the same physical location. Forexample, the delivery and/or dispersal processes may be performed undera first set of conditions while the open substrate is retained in afirst physical location, and the detection process may be performedunder a second set of conditions while the open substrate is retained inthe same physical location. Alternatively, the first environment maycomprise a first physical location in which the open substrate isaccessible to a rotational unit configured to rotate the open substrateduring the delivery and/or dispersal processes. The second environmentmay comprise a second physical location in which the open substrate isaccessible to the detector system. As noted above, one or morecomponents of the detector system and/or the open substrate may be inmotion during the detection process. The second physical location maycomprise a mechanism for supporting the open substrate while retainingit in a stationary state as well as a mechanism (e.g., a rotationalunit) for moving the detector system or a component thereof relative tothe open substrate (e.g., as described herein). The first and secondenvironments may in physical proximity to one another. In an example,the first environment may be disposed in a first physical location of anapparatus that is located above a second physical location of theapparatus that is part of the second environment. In another example,the first environment may be disposed in a first physical location of anapparatus that is located adjacent or somewhat adjacent to a secondphysical location of the apparatus that is part of the secondenvironment. The first and second environments may be separable by oneor more barriers. In an example, a retractable barrier such as a slidingdoor separates the first and second environments. The retractablebarrier may remain in a closed state during delivery and/or dispersalprocesses and may then be retracted to permit translation of the opensubstrate from the first environment to the second environment forsubsequent detection. The retractable barrier may be retained in aclosed state during the detection process. The open substrate may beretained in a container, which container is transferred with the opensubstrate between the first and second environments.

The first and second environments may comprise one or more differentoperating conditions. For example, the first environment may comprise afirst temperature, humidity, and pressure and the second environment maycomprise a second temperature, humidity, and pressure, where at leastone of temperature, humidity, and pressure differ between the first andsecond environments. A given environment may comprise multipletemperature, humidity, and/or pressure zones, and one or more such zonesmay differ in the first and second environments.

The present disclosure also provides apparatus and computer readablemedia for implementing the methods provided herein. For example, thepresent disclosure provides a computer-readable medium comprisingnon-transitory instructions stored thereon, which when executed causeone or more computer processors to implement the methods providedherein. The present disclosure also provides an apparatus for analytedetection or analysis comprising a housing configured to receive an opensubstrate having an array of immobilized analytes thereon (e.g., asdescribed herein). The apparatus may comprise one or more dispensersconfigured to deliver a solution having a plurality of probes to aregion proximal to a central axis of the open substrate. The apparatusmay also comprise a rotational unit configured to rotate the opensubstrate about the central axis to disperse the solution across theopen substrate at least by centrifugal force, such that at least oneprobe of the plurality of probes binds to at least one immobilizedanalyte of the immobilized analytes to form a bound probe. Therotational unit may be disposed in a first area of the apparatus, whichfirst area is distinct from a second area of the apparatus. Theapparatus may also comprise a detector system, which detector system maycomprise a sensor (e.g., line-scan camera) and an illumination source(e.g., as described herein). The detector system may be disposed in thesecond area of the apparatus. Alternatively, the detector system may bedisposed in the first area of the apparatus. The open substrate maycomprise a first area and a second area, where the first area and thesecond area comprise subsets of the array of immobilized analytes, areat different radial positions of the open substrate with respect to thecentral axis and are spatially resolved by the detector system. Thebound probe may be disposed in the first area of the open substrate, andthe detector system may be programmed to perform a non-linear scan ofthe open substrate and detect at least one signal from the bound probeat the first area of the open substrate. The apparatus may comprise oneor more processors configured to, for example, direct dispersal anddelivery of one or more solutions to the open substrate or direct thedetector system to detect one or more signals from the open substrate.The processor may be programmed to direct the detector system tocompensate for velocity differences at different radial positions of thearray with respect to the central axis of the open substrate within ascanned area. For example, the processor may be programmed to direct thedetector system to scan two or more regions of the open substrate at twoor more different scan rates, respectively, to at least partiallycompensate for tangential velocity differences in the two or moreregions. The apparatus may further comprise one or more optics, such asone or more optics that are configured to generate an anamorphicmagnification gradient that is, e.g., substantially transverse to ascanning direction along the open substrate (e.g., as described herein).A processor may be programmed to adjust the gradient to compensate fordifferent imaged radial positions with respect to the central axis ofthe open substrate.

System Architectures for High-Throughput Processing

The nucleic acid sequencing systems and optical systems described herein(or any elements thereof) may be combined in a variety of architectures.

FIG. 23A shows an architecture for a system 1200 a comprising astationary substrate and moving fluidics and optics. The system 1200 amay comprise substrate 310 described herein. The substrate may beconfigured to rotate, as described herein. The substrate may be adheredor otherwise affixed to a chuck (not shown in FIG. 23A), as describedherein. The system may further comprise fluid channel 330 and fluidoutlet port 335 described herein, and/or any other fluid channel andfluid outlet port described herein. The fluid channel and fluid outletport may be configured to dispense any solution described herein. Thefluid channel and fluid outlet port may be configured to move 1215 arelative to the substrate. For instance, the fluid channel and fluidoutlet port may be configured to move to a position above (such as nearthe center of) the substrate during periods of time in which the fluidchannel and fluid outlet port are dispensing a solution. The fluidchannel and fluid outlet port may be configured to move to a positionaway from the substrate during the period in which the fluid channel andfluid outlet port are not dispensing a solution. Alternatively, thereverse may apply. The system may further comprise optical imagingobjective 1110 described herein. The optical imaging objective may beconfigured to move 1210 a relative for the substrate. For instance, theoptical imaging objective may be configured to move to a position above(such as near the center of) the substrate during periods of time inwhich the substrate is being imaged. The optical imaging objective maybe configured to move to a position away from the substrate during theperiod in which the substrate is not being imaged. The system mayalternate between dispensing of solutions and imaging, allowing rapidsequencing of the nucleic acids attached to the substrate using thesystems and methods described herein.

FIG. 23B shows an architecture for a system 1200 b comprising a movingsubstrate and stationary fluidics and optics. The system 1200 b maycomprise substrate 310 described herein. The substrate may be configuredto rotate, as described herein. The substrate may be adhered orotherwise affixed to a chuck (not shown in FIG. 23B), as describedherein. The system may further comprise fluid channel 330 and fluidoutlet port 335 described herein, or any other fluid channel and fluidoutlet port described herein. The fluid channel and fluid outlet portmay be configured to dispense any solution described herein. The systemmay further comprise optical imaging objective 1110 described herein.The fluid channel, fluid outlet port, and optical imaging objective maybe stationary. The substrate may be configured to move 1210 b relativeto the fluid channel, fluid outlet port, and optical imaging objective.For instance, the substrate may be configured to move to a position suchthat the fluid channel and fluid outlet port are above (such as near thecenter of) the substrate during periods of time in which the fluidchannel and fluid outlet port are dispensing a solution. The substratemay be configured to move to a position away from the fluid channel andfluid outlet port during the period in which the fluid channel and fluidoutlet port are not dispensing a solution. The substrate may beconfigured to radially scan the objective over the substrate duringperiods of time in which the substrate is being imaged. The substratemay be configured to move to a position away from the optical imagingobjective during the period in which the substrate is not being imaged.The system may alternate between dispensing of solutions and imaging,allowing rapid sequencing of the nucleic acids attached to the substrateusing the systems and methods described herein.

FIG. 23C shows an architecture for a system 1200 c comprising aplurality of stationary substrates and moving fluidics and optics. Thesystem 1200 c may comprise first and second substrates 310 a and 310 b.The first and second substrates may be similar to substrate 310described herein. The first and second substrates may be configured torotate, as described herein. The first and second substrates may beadhered or otherwise affixed to first and second chucks (not shown inFIG. 23C), as described herein. The system may further comprise firstfluid channel 330 a and first fluid outlet port 335 a. First fluidchannel 330 a may be similar to fluid channel 330 described herein orany other fluid channel described herein. First fluid outlet port 335 amay be similar to fluid outlet port 335 described herein or any otherfluid outlet port described herein. The system may further comprisesecond fluid channel 330 b and second fluid outlet port 335 b. Secondfluid channel 330 b may be similar to fluid channel 330 described hereinor any other fluid channel described herein. Second fluid outlet port335 a may be similar to fluid outlet port 335 described herein or anyother fluid outlet port described herein. The first fluid channel andfirst fluid outlet port may be configured to dispense any solutiondescribed herein. The second fluid channel and second fluid outlet portmay be configured to dispense any solution described herein.

The system may further comprise optical imaging objective 1110. Opticalimaging objective 1110 may be configured to move 1210 c relative to thefirst and second substrates. For instance, the optical imaging objectivemay be configured to move to a position above (such as near the centerof, or radially scanning) the first substrate during periods of time inwhich the first fluid channel and first fluid outlet port are notdispensing a solution to the second substrate (and during which thefirst substrate is to be imaged). The optical imaging objective may beconfigured to move to a position away from the first substrate duringthe period in which the first fluid channel and first fluid outlet portare dispensing a solution. The optical imaging objective may beconfigured to move to a position above (such as near the center of, orradially scanning) the second substrate during periods of time in whichthe second fluid channel and second fluid outlet port are not dispensinga solution to the second substrate (and during which the secondsubstrate is to be imaged). The optical imaging objective may beconfigured to move to a position away from the second substrate duringthe period in which the second fluid channel and second fluid outletport are dispensing a solution.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the optical imaging objective maybe moved from the second substrate to the first substrate. A solutionmay then be dispensed to the second substrate during a period of time inwhich the first substrate is being imaged. This alternating pattern ofdispensing and imaging may be repeated, allowing rapid sequencing of thenucleic acids attached to the first and second substrates using thesystems and methods described herein. The alternating pattern ofdispensing and imaging may speed up the sequencing by increasing theduty cycle of the imaging process or the solution dispensing process.

Though depicted as comprising two substrates, two fluid channels, twofluid outlet ports, and one optical imaging objective in FIG. 23C,system 1200 c may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and/or at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. Each optical imagingobjective may be moved between substrates as described herein.

FIG. 23D shows an architecture for a system 1200 d comprising aplurality of moving substrates on a rotary stage and stationary fluidicsand optics. The system 1200 d may comprise first and second substrates310 a and 310 b. The first and second substrates may be similar tosubstrate 310 described herein. The first and second substrates may beconfigured to rotate, as described herein. The first and secondsubstrates may be adhered or otherwise affixed to first and secondchucks (not shown in FIG. 23D), as described herein. The first andsecond substrates may be affixed to a rotating stage 1220 d (such asapproximately at opposing ends of the rotating stage). The rotatingstage may be configured to rotate about an axis. The axis may be an axisthrough the center of the substrate. The axis may be an off-center axis.The rotating stage may approximately scan the radius of the substrate310 b. The system may further comprise fluid channel 330 and fluidoutlet port 335. The fluid channel and fluid outlet port may beconfigured to dispense any solution described herein. The system mayfurther comprise optical imaging objective 1110. A longitudinal axis ofthe imaging objective 1110 may not be coincident with a central axis ofthe second substrate 310 b (although this is difficult to distinguish inFIG. 23D). The imaging objective 1110 may be positioned at some distancefrom a center of the second substrate 310 b.

The rotating stage may be configured to alter the relative positions ofthe first and second substrates to carry out different sequencingoperations. For instance, the rotating stage may be configured to rotatesuch that the optical imaging objective is in a position above or inoptical communication with the first substrate during periods of time inwhich the fluid channel and fluid outlet port are not dispensing asolution to the first substrate (and during which the first substrate isto be imaged). The rotating stage may be configured to rotate such thatthe optical imaging objective is away from the first substrate duringthe period in which the fluid channel and fluid outlet port aredispensing a solution to the first substrate. The rotating stage may beconfigured to rotate such that the optical imaging objective is in aposition above or in optical communication with the second substrateduring periods of time in which the fluid channel and fluid outlet portare not dispensing a solution to the second substrate (and during whichthe second substrate is to be imaged). The rotating stage may beconfigured to rotate such that the optical imaging objective is awayfrom the second substrate during the period in which the fluid channeland fluid outlet port are dispensing a solution to the second substrate.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the rotating stage may be rotatedsuch that a solution may be dispensed to the second substrate during aperiod of time in which the first substrate is being imaged. Thisalternating pattern of dispensing and imaging may be repeated, allowingrapid sequencing of the nucleic acids attached to the first and secondsubstrates using the systems and methods described herein. Thealternating pattern of dispensing and imaging may speed up thesequencing by increasing the duty cycle of the imaging process or thesolution dispensing process.

Though depicted as comprising two substrates, one fluid channel, onefluid outlet port, and one optical imaging objective in FIG. 23D, system1200 d may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The rotating stagemay be rotated to place any substrate under any fluid channel, fluidoutlet port, or optical imaging objective at any time.

FIG. 23E shows an architecture for a system 1200 e comprising aplurality of stationary substrates and moving optics. The system 1200 dmay comprise first and second substrates 310 a and 310 b. The first andsecond substrates may be similar to substrate 310 described herein. Thefirst and second substrates may be configured to rotate, as describedherein. The first and second substrates may be adhered or otherwiseaffixed to first and second chucks (not shown in FIG. 23E), as describedherein. The system may further comprise first fluid channel 330 a andfirst fluid outlet port 335 a. First fluid channel 330 a may be similarto fluid channel 330 described herein or any other fluid channeldescribed herein. First fluid outlet port 335 a may be similar to fluidoutlet port 335 described herein or any other fluid outlet portdescribed herein. The first fluid channel and first fluid outlet portmay be configured to dispense any solution described herein. The systemmay further comprise second fluid channel 330 b and second fluid outletport 335 b. Second fluid channel 330 b may be similar to fluid channel330 described herein or any other fluid channel described herein. Secondfluid outlet port 335 b may be similar to fluid outlet port 335described herein or any other fluid outlet port described herein. Thesecond fluid channel and second fluid outlet port may be configured todispense any solution described herein.

The system may further comprise optical imaging objective 1110. Theoptical imaging objective may be attached to an imaging arm 1230 e. Theoptical imaging objective may be configured to move 1220 e along theoptical imaging arm to image an entire area of the first or secondsubstrate. The optical imaging arm may be configured to rotate 1210 e.The optical imaging arm may be configured to rotate such that theoptical imaging objective is in a position above or in opticalcommunication with the first substrate during periods of time in whichthe first fluid channel and first fluid outlet port are not dispensing asolution to the first substrate (and during which the first substrate isto be imaged). The optical imaging arm may be configured to rotate suchthat the optical imaging objective is away from the first substrateduring the period in which the first fluid channel and first fluidoutlet port are dispensing a solution to the first substrate. Theoptical imaging arm may be configured to rotate such that the opticalimaging objective is in a position above or in optical communicationwith the second substrate during periods of time in which the secondfluid channel and second fluid outlet port are not dispensing a solutionto the second substrate (and during which the second substrate is to beimaged). The optical imaging arm may be configured to rotate such thatthe optical imaging objective is away from the second substrate duringthe period in which the second fluid channel and second fluid outletport are dispensing a solution to the second substrate.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the optical imaging arm may berotated such that a solution may be dispensed to the second substrateduring a period of time in which the first substrate is being imaged.This alternating pattern of dispensing and imaging may be repeated,allowing rapid sequencing of the nucleic acids attached to the first andsecond substrates using the systems and methods described herein. Thealternating pattern of dispensing and imaging may speed up thesequencing by increasing the duty cycle of the imaging process or thesolution dispensing process.

Though depicted as comprising two substrates, two fluid channels, twofluid outlet ports, and one optical imaging objective in FIG. 23E,system 1200 e may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The optical imagingarm may be rotated to place any substrate under any fluid channel, fluidoutlet port, or optical imaging objective at any time.

FIG. 23F shows an architecture for a system 1200 f comprising aplurality of moving substrates and stationary fluidics and optics. Thesystem 1200 f may comprise first and second substrates 310 a and 310 b.The first and second substrates may be similar to substrate 310described herein. The first and second substrates may be configured torotate, as described herein. The first and second substrates may beadhered or otherwise affixed to first and second chucks (not shown inFIG. 23F), as described herein. The first and second substrates may beaffixed to opposing ends of a moving stage 1220 f. The moving stage maybe configured to move 1210 f. The system may further comprise firstfluid channel 330 a and first fluid outlet port 335 a. First fluidchannel 330 a may be similar to fluid channel 330 described herein orany other fluid channel described herein. First fluid outlet port 335 amay be similar to fluid outlet port 335 described herein or any otherfluid outlet port described herein. The first fluid channel and firstfluid outlet port may be configured to dispense any solution describedherein. The system may further comprise second fluid channel 330 b andsecond fluid outlet port 335 b. Second fluid channel 330 b may besimilar to fluid channel 330 described herein or any other fluid channeldescribed herein. Second fluid outlet port 335 b may be similar to fluidoutlet port 335 described herein or any other fluid outlet portdescribed herein. The second fluid channel and second fluid outlet portmay be configured to dispense any solution described herein. The systemmay further comprise optical imaging objective 1110.

The moving stage may be configured to move such that the optical imagingobjective is in a position above or in optical communication with thefirst substrate during periods of time in which the first fluid channeland first fluid outlet port are not dispensing a solution to the firstsubstrate (and during which the first substrate is to be imaged). Themoving stage may be configured to move such that the optical imagingobjective is away from the first substrate during the period in whichthe first fluid channel and first fluid outlet port are dispensing asolution to the first substrate. The moving stage may be configured tomove such that the optical imaging objective is in a position above orin optical communication with the second substrate during periods oftime in which the second fluid channel and second fluid outlet port arenot dispensing a solution to the second substrate (and during which thesecond substrate is to be imaged). The moving stage may be configured tomove such that the optical imaging objective is away from the secondsubstrate during the period in which the second fluid channel and secondfluid outlet port are dispensing a solution to the second substrate.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the moving stage may move suchthat a solution may be dispensed to the second substrate during a periodof time in which the first substrate is being imaged. This alternatingpattern of dispensing and imaging may be repeated, allowing rapidsequencing of the nucleic acids attached to the first and secondsubstrates using the systems and methods described herein. Thealternating pattern of dispensing and imaging may speed up thesequencing by increasing the duty cycle of the imaging process or thesolution dispensing process.

Though depicted as comprising two substrates, two fluid channels, twofluid outlet ports, and one optical imaging objective in FIG. 23F,system 1200 f may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The moving stage maymove so as to place any substrate under any fluid channel, fluid outletport, or optical imaging objective at any time.

FIG. 23G shows an architecture for a system 2300 g comprising aplurality of moving substrates and stationary fluidics and optics. Thesystem 2300 g may comprise first and second substrates 310 a and 310 b.The first and second substrates may be similar to substrate 310described herein. The first and second substrates may be configured torotate, as described herein. The first and second substrates may beadhered or otherwise affixed to first and second chucks (not shown inFIG. 23G), as described herein. The first and second substrates may beconfigured to translate along a stationary stage 2320 g. The first andsecond substrates may be configured to move between a first fluidstation comprising a first fluid channel 330 a and first fluid outletport 335 a, a second fluid station comprising a second fluid channel 330b and second fluid outlet port 335 b, and an imaging station comprisingan optical imaging objective 1110. FIG. 23G illustrates a configurationwhere the first substrate is positioned at the first fluid station andthe second substrate is positioned at the imaging station. In anotherconfiguration, the first and second substrates may undergo relativetranslation with respect to the optical head so that the first substrateis positioned at the imaging station and the second substrate ispositioned at the second fluid station. The first and second translatingsubstrates may be configured to move such that the optical imagingobjective is in a position above or in optical communication with thefirst substrate during periods of time in which the first fluid channeland first fluid outlet port are not dispensing a solution to the firstsubstrate (and during which the first substrate is to be imaged). Thefirst and second translating substrates may be configured to move suchthat the optical imaging objective is away from the first substrateduring the period in which the first fluid channel and first fluidoutlet port are dispensing a solution to the first substrate. The firstand second translating substrates may be configured to move such thatthe optical imaging objective is in a position above or in opticalcommunication with the second substrate during periods of time in whichthe second fluid channel and second fluid outlet port are not dispensinga solution to the second substrate (and during which the secondsubstrate is to be imaged). The first and second translating substratesmay be configured to move such that the optical imaging objective isaway from the second substrate during the period in which the secondfluid channel and second fluid outlet port are dispensing a solution tothe second substrate.

The timing of dispensing of a solution and imaging of a substrate may besynchronized. For instance, a solution may be dispensed to the firstsubstrate during a period of time in which the second substrate is beingimaged. Once the solution has been dispensed to the first substrate andthe second substrate has been imaged, the moving stage may move suchthat a solution may be dispensed to the second substrate during a periodof time in which the first substrate is being imaged. This alternatingpattern of dispensing and imaging may be repeated, allowing rapidsequencing of the nucleic acids attached to the first and secondsubstrates using the systems and methods described herein. Thealternating pattern of dispensing and imaging may speed up thesequencing by increasing the duty cycle of the imaging process or thesolution dispensing process.

Though depicted as comprising two substrates, two fluid channels, twofluid outlet ports, and one optical imaging objective in FIG. 23G,system 2300 g may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The translatingsubstrates may move so as to place any substrate under any fluidchannel, fluid outlet port, or optical imaging objective at any time.

FIG. 23H shows an architecture for a system 1200 g comprising aplurality of substrates moved between a plurality of processing bays.The system 1200 g may comprise first, second, third, and fourthsubstrates 310 a, 310 b, 310 c, 310 d, and 310 e, respectively. Thefirst, second, third, fourth, and fifth substrates may be similar tosubstrate 310 described herein. The first, second, third, fourth, andfifth substrates may be configured to rotate, as described herein. Thefirst, second, third, fourth, and fifth substrates may be adhered orotherwise affixed to first, second, third, fourth, and fifth chucks (notshown in FIG. 23H), respectively, as described herein.

The system may further comprise first fluid channel 330 a and firstfluid outlet port 335 a. First fluid channel 330 a may be similar tofluid channel 330 described herein or any other fluid channel describedherein. First fluid outlet port 335 a may be similar to fluid outletport 335 described herein or any other fluid outlet port describedherein. The first fluid channel and first fluid outlet port may beconfigured to dispense any solution described herein. The first fluidchannel and first fluid outlet port may be regarded as a firstprocessing bay. The first processing bay may be configured to perform afirst processing operation, such as dispensing of a first solution toany of the first, second, third, fourth, or fifth substrates.

The system may further comprise second fluid channel 330 b and secondfluid outlet port 335 b. Second fluid channel 330 b may be similar tofluid channel 330 described herein or any other fluid channel describedherein. Second fluid outlet port 335 b may be similar to fluid outletport 335 described herein or any other fluid outlet port describedherein. The second fluid channel and second fluid outlet port may beconfigured to dispense any solution described herein. The second fluidchannel and second fluid outlet port may be regarded as a secondprocessing bay or processing station. The second processing bay may beconfigured to perform a second processing operation, such as dispensingof a second solution to any of the first, second, third, fourth, orfifth substrates.

The system may further comprise third fluid channel 330 c and thirdfluid outlet port 335 c. Third fluid channel 330 c may be similar tofluid channel 330 described herein or any other fluid channel describedherein. Third fluid outlet port 335 c may be similar to fluid outletport 335 described herein or any other fluid outlet port describedherein. The third fluid channel and third fluid outlet port may beconfigured to dispense any solution described herein. The third fluidchannel and third fluid outlet port may be regarded as a thirdprocessing bay or processing station. The third processing bay may beconfigured to perform a third processing operation, such as dispensingof a third solution to any of the first, second, third, fourth, or fifthsubstrates.

The system may further comprise fourth fluid channel 330 d and fourthfluid outlet port 335 d. Fourth fluid channel 330 d may be similar tofluid channel 330 described herein or any other fluid channel describedherein. Fourth fluid outlet port 335 d may be similar to fluid outletport 335 described herein or any other fluid outlet port describedherein. The fourth fluid channel and fourth fluid outlet port may beconfigured to dispense any solution described herein. The fourth fluidchannel and fourth fluid outlet port may be regarded as a fourthprocessing bay or processing station. The fourth processing bay may beconfigured to perform a fourth processing operation, such as dispensingof a fourth solution to any of the first, second, third, fourth, orfifth substrates.

The system may further comprise a scanning optical imaging objective1110. The optical imaging objective may be regarded as a fifthprocessing bay or processing station.

The system may further comprise a moving arm 1220 g. The moving arm maybe configured to move laterally 1210 g or rotate 1215 g. The moving armmay be configured to move any of the first, second, third, fourth, orfifth substrates between different processing stations (such as bypicking up substrates and moving them to new locations). For instance,at a first point in time, the first substrate may undergo a firstoperation (such as dispensing of a first solution) at the firstprocessing bay, the second substrate may undergo a second operation(such as dispensing of a second solution) at the second processing bay,the third substrate may undergo a third operation (such as dispensing ofa third solution) at the first processing bay, the fourth substrate mayundergo a fourth operation (such as dispensing of a fourth solution) atthe fourth processing bay, and the fifth substrate may be imaged at thefifth processing bay. Upon completion of one or more of the first,second, third, or fourth operations, or of imaging, the moving arm maymove one or more of the first, second, third, fourth, or fifthsubstrates to one or more of the first, second, third, fourth, or fifthprocessing bays, where another operation may be completed. The patternof completing one or more operations and moving one or more substratesto another processing bay to complete another operation may be repeated,allowing rapid sequencing of the nucleic acids attached to the first,second, third, fourth, and fifth substrates using the systems andmethods described herein. The alternating pattern of dispensing andimaging may speed up the sequencing by increasing the duty cycle of theimaging process or the solution dispensing process.

Though depicted as comprising five substrates, four fluid channels, fourfluid outlet ports, and one optical imaging objective in FIG. 23H,system 1200 g may comprise any number of each of the substrates, fluidchannels, fluid outlet ports, and optical imaging objectives. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. Each substrate may be adhered or otherwiseaffixed to a chuck as described herein. The system may comprise at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, or at least 10 fluid channels and at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 fluid outlet ports. Each fluidchannel and fluid outlet port may be configured to dispense a solutionas described herein. The system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 optical imaging objectives. The moving arm maymove so as to place any substrate under any fluid channel, fluid outletport, or optical imaging objective at any time.

FIG. 23I shows an architecture for a system 1200 h comprising aplurality of imaging heads scanning with shared translation androtational axes and independently rotating fields. The system maycomprise first and second read heads 1005 and 1015, respectively,configured to image substrate 310. The first and second read heads maybe similar to any read head described herein (such as with respect toFIG. 14). At a particular point in time, the first and second read headsmay be configured to image first and second paths 1010 and 1020,respectively. The first and second paths may be similar to any pathsdescribed herein (such as with respect to FIG. 14). The first and secondread heads may be configured to move 1210 h in a substantially radialdirection over the spinning substrate, thereby scanning the substrate.In the event that either the first or second read head does not moveprecisely radially, an image field or sensor of the read head may rotateto maintain a substantially tangential scan direction, as described withrespect to FIG. 34. A field rotation may be accomplished using rotatingprisms. Alternatively or in addition, mirrors or other optical elementsmay be used.

Though depicted as comprising two read heads and two imaging paths inFIG. 23I, system 1200 h may comprise any number of read heads or imagingpaths. For instance, the system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 read heads. The system may comprise at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 imaging paths.

FIG. 23J shows an architecture for a system 2300 j comprising aplurality of imaging heads scanning with shared translation androtational axes and independently rotating fields. The system maycomprise first, second, third, and fourth read heads 1005, 1015, 1025,and 1035, respectively, configured to image substrate 310. The first,second, third, and fourth read heads may be similar to any read headdescribed herein (such as with respect to FIG. 14). At a particularpoint in time, the first, second, third, and fourth read heads may beconfigured to image first, second, third, and fourth paths 1010, 1020,1030, and 1040, respectively. The first, second, third, and fourth pathsmay be similar to any paths described herein (such as with respect toFIG. 14). The first, second, third, and fourth read heads may beconfigured to move 1210 h in a substantially radial direction over thespinning substrate, thereby scanning the substrate. In the event thatthe first, second, third, and fourth read head does not move preciselyradially, an image field or sensor of the read head may rotate tomaintain a substantially tangential scan direction. A field rotation maybe accomplished using rotating prisms. Alternatively or in addition,mirrors or other optical elements may be used.

Though depicted as comprising four read heads and four imaging paths inFIG. 23J, system 2300 j may comprise any number of read heads or imagingpaths. For instance, the system may comprise at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10 read heads. The system may comprise at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10 imaging paths.

FIG. 23K shows an architecture for a system 1200 i comprising multiplespindles scanning with a shared optical detection system. The system maycomprise first and second substrates 310 a and 310 b, respectively. Thefirst and second substrates may be similar to substrate 310 describedherein. The first and second substrates may be affixed to first andsecond spindles, respectively. The first and second spindles may impartrotational motion to the first and second substrates, respectively. Thesystem may comprise first and second optical imaging objectives 1110 aand 1110 b, respectively. The first and second optical imagingobjectives may be similar to optical imaging objective 1110 describedherein. The first and second optical imaging objectives may beconfigured to collect light from the first and second substrates,respectively. The first and second optical imaging objectives may passlight collected from the first and second substrates, respectively, tofirst and second mirrors 1280 a and 1280 b, respectively. In some cases,only one of the first and second optical imaging objective will collectlight at a particular instance in time.

The first and second mirrors may pass the light to a shared movablemirror. When in a first configuration 1285 a, the shared movable mirrormay direct light from the first substrate to a beamsplitter 1295. Thebeamsplitter may comprise a dichroic mirror. For example, as illustratedin FIG. 23K, the beamsplitter may be configured to reflect an excitationlight from an excitation light source 1290 toward a substrate andtransmit the light from a substrate toward the detector 370. In analternative configuration (not shown in FIG. 23K), the beamsplitter maybe configured to transmit an excitation light from an excitation lightsource 1290 toward the substrate and reflect the light from a substratetoward the detector 370. The beamsplitter may pass or reflect light to adetector 370, allowing the first substrate to be imaged. The firstsubstrate may be configured to be translated 1210 i, allowing differentlocations on the first substrate to be imaged.

When in a second configuration 1285 b, the shared movable mirror maydirect light from the second substrate to the beamsplitter 1295. Thebeamsplitter may pass or reflect light to a detector 370, allowing thesecond substrate to be imaged. The second substrate may be configured tobe translated 1210 i, allowing different locations on the secondsubstrate to be imaged. Thus, by moving the movable mirror, the firstand second substrates may be imaged by a shared optical system.

The system may further comprise an excitation light source 1290. Thelight source may be configured to provide excitation light (such as forfluorescence imaging) to the first or second substrate. The excitationlight may be selectively delivered to the first or second substrateusing the movable mirror in a similar manner as for detection describedherein.

Though depicted as comprising two substrates, two imaging opticalobjectives, and two mirrors in FIG. 23K, system 1200 i may comprise anynumber of substrates, imaging optical objectives, or mirrors. Forinstance, the system may comprise at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10 substrates. The system may comprise at least 1, at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, or at least 10 imaging optical objectives. The system maycomprise at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, or at least 10 mirrors.

FIG. 23I shows an architecture for a system comprising a plurality ofimaging heads scanning with shared translation and rotational axes andindependently rotating fields.

FIG. 23K shows an architecture for a system comprising multiple spindlesscanning with a shared optical detection system.

FIG. 24 shows an architecture for a system 1300 comprising a pluralityof rotating spindles. The system 1300 may comprise substrate 310described herein. The substrate may be configured to rotate, asdescribed herein. The system may further comprise fluid channel 330 andfluid outlet port 335 described herein, or any other fluid channel andfluid outlet port described herein. The fluid channel and fluid outletport may be configured to dispense any solution described herein. Thefluid channel and fluid outlet port may be configured to move 1315 arelative to the substrate. For instance, the fluid channel and fluidoutlet port may be configured to move to a position above (such as nearthe center of) the substrate during periods of time in which the fluidchannel and fluid outlet port are dispensing a solution. The fluidchannel and fluid outlet port may be configured to move to a positionaway from the substrate during the period in which the fluid channel andfluid outlet port are not dispensing a solution. The system may furthercomprise optical imaging objective 1110 described herein. The opticalimaging objective may be configured to move 1310 a relative for thesubstrate. For instance, the optical imaging objective may be configuredto move to a position above (such as near the center of, or radiallyscanning) the substrate during periods of time in which the substrate isbeing imaged. The optical imaging objective may be configured to move toa position away from the substrate during the period in which thesubstrate is not being imaged.

The system may further comprise a first spindle 1305 a and a secondspindle 1305 b. The first spindle may be interior to the second spindle.The first spindle may be exterior to the second spindle. The secondspindle may be interior to the first spindle. The second spindle may beexterior to the first spindle. The first and second spindles may each beconfigured to rotate independently of each other. The first and secondspindles may be configured to rotate with different angular velocities.For instance, the first spindle may be configured to rotate with a firstangular velocity and the second spindle may be configured to rotate witha second angular velocity. The first angular velocity may be less thanthe second angular velocity. The first spindle may be configured torotate at a relatively low angular velocity (such as an angular velocitybetween about 0 rpm and about 100 rpm) during periods in which asolution is being dispensed to the substrate. The second spindle may beconfigured to rotate at a relatively high angular velocity (such as anangular velocity between about 100 rpm and about 1,000 rpm) duringperiods in which the substrate is being imaged. Alternatively, thereverse may apply. The substrate may be transferred between the firstand second spindles to complete each of the dispensing and imagingoperations.

The system may comprise any number of spindles. For example, the systemmay comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or morespindles. Alternatively or in addition, the system may comprise at mostabout 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 spindle. A given spindle maybe interior or exterior relative to one or more other spindles in thesystem. In some instances, each of the spindles may rotate independentlyof each other. In some instances, at least a subset of the spindles mayrotate independently of each other. In some instances, at least a subsetof the spindles may rotate dependently of each other (e.g.,simultaneously at the same angular velocity). The spindles may rotatewith respect to the same axis or different axes. In some instances, eachspindle may rotate with different angular velocities. In some instances,at least a subset of the spindles may rotate with different angularvelocities.

Though depicted as utilizing a moving fluid channel and optical imagingobjective in FIG. 24, the system 1300 may be configured in other mannersas described herein. For instance, the system may be configured suchthat the fluid channel and optical imaging objective are stationary, andthe substrate is configured to move. The system may be configured in anyother manner described herein.

Application to Other Analytes

Though described herein as useful for sequencing nucleic acids, thesystems and method described herein may be applied to other analytesand/or other applications processing such analytes. FIG. 25 shows aflowchart for an example of a method 1400 for processing an analyte.

In a first operation 1410, the method may comprise providing a substratecomprising a planar array having immobilized thereto an analyte, whereinthe substrate is configured to rotate with respect to an axis. The axismay be an axis through the center of the substrate. The axis may be anoff-center axis. The substrate may be any substrate described herein. Insome instances, the planar array may comprise a single type of analyte.In other instances, the planar array may comprise two or more types ofanalytes. The two or more types of analytes may be arranged randomly.The two or more types of analytes may be arranged in a regular pattern.For example, two types of analytes may be arranged in a radiallyalternating pattern. The analyte may be any biological sample describedherein or derivative thereof. For example, the analyte may be a singlecell analyte. The analyte may be a nucleic acid molecule. The analytemay be a protein molecule. The analyte may be a single cell. The analytemay be a particle. The analyte may be an organism. The analyte may bepart of a colony. In some cases, the analyte may be or be derived from anon-biological sample. The analyte may be immobilized in an individuallyaddressable location on the planar array. The analyte may be immobilizedto the substrate via a linker configured to bind to the analyte. Forexample, the linker may comprise a carbohydrate molecule. The link maycomprise an affinity binding protein. The linker may be hydrophilic. Thelinker may be hydrophobic. The linker may be electrostatic. The linkermay be labeled. The linker may be integral to the substrate. The linkermay be an independent layer on the substrate.

In a second operation 1420, the method may comprise directing a solutioncomprising a plurality of reactants across the planar array duringrotation of the substrate. The solution may comprise any solution orreagent described herein. The plurality of reactants may be configuredto interact with the analyte immobilized to the planar array. Forexample, where the analyte is a nucleic acid molecule, the plurality ofreactants may comprise a plurality of probes. A given probe of theplurality of probes may comprise a random sequence or a targetedsequence, such as a homopolymer sequence or a dibase or tribaserepeating sequence. In some instances, the probe may be a dibase probe.In some instances, the probe may be about 1 to 10 bases in length. Insome instances, the probe may be about 10 to 20 bases in length. In someinstances, the probe may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, or more bases.Alternatively or in combination the probe may be at most about 50, 40,30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,1 base. In another example, where the analyte is a protein molecule, theplurality of reactants may comprise a plurality of antibodies. A givenantibody of the plurality of antibodies may have binding specificity toone or more types of proteins. In other instances, the plurality ofreactants may comprise any combination of a plurality of oligonucleotidemolecules, carbohydrate molecules, lipid molecules, affinity bindingproteins, aptamers, antibodies, enzymes, or other reagents. Theplurality of reactants may be hydrophilic. The plurality of reactantsmay be hydrophobic. The plurality of reactants may be electrostatic. Theplurality of reactants may be labeled. The plurality of reactants maycomprise a mixture of labeled and unlabeled components. In someinstances, the plurality of reactants may not be labeled.

In an operation 1430, the method may comprise subjecting the analyte toconditions sufficient to cause a reaction or interaction between theanalyte and the plurality of reactants. In an operation 1440, the methodmay comprise detecting a signal indicative of the reaction between theanalyte and the plurality of reactants, thereby analyzing the analyte.In some cases, a reactant may undergo a reaction with the analyte.Alternatively or in addition, the reactant may bind to or interact withthe analyte. One or more of the analyte or the reactant may undergo aconformational change, chemical change, state change, or any combinationthereof upon interaction with the analyte.

The method may further comprise, prior to operation 1410, directing theanalyte across the substrate comprising the linker. For example, priorto or during dispensing of the analyte, the substrate may be rotated tocoat the substrate surface and/or the planar array with the analyte. Insome instances, the analyte may be coupled to a bead, which bead isimmobilized to the planar array.

The method may further comprise recycling, as described elsewhereherein, a subset of the solution that has contacted the substrate. Therecycling may comprise collecting, filtering, and reusing the subset ofthe solution. The filtering may comprise molecular filtering. Themolecular filtering may comprise specific nucleic acid filtering (i.e.filtering for a specific nucleic acid). The nucleic acid filtering maycomprise exposure of the solution to an array of oligonucleotideextension compounds which may specifically bind to contaminantnucleotides or nucleic acids.

The signal may be an optical signal. The signal may be a fluorescencesignal. The signal may be a light absorption signal. The signal may be alight scattering signal. The signal may be a luminescent signal. Thesignal may be a phosphorescence signal. The signal may be an electricalsignal. The signal may be an acoustic signal. The signal may be amagnetic signal. The signal may be any detectable signal. Alternativelyor in addition to the optical sensors described herein, the system maycomprise one or more other detectors (e.g., acoustic detector, etc.)configured to detect the detectable signal.

In some instances, the method may further comprise, prior to operation1420, subjecting the substrate to rotation with respect to the centralaxis.

In some instances, the method may further comprise terminating rotationof the substrate prior to detecting the signal in operation 1440. Inother instances, the signal may be detected in operation 1440 while thesubstrate is rotating.

The signal may be generated by binding of a label to the analyte. Thelabel may be bound to a molecule, particle, cell, or organism. The labelmay be bound to the molecule, particle, cell, or organism prior tooperation 1410. The label may be bound to the molecule, particle, cell,or organism subsequent to operation 1410. The signal may be generated byformation of a detectable product by a chemical reaction. The reactionmay comprise an enzymatic reaction. The signal may be generated byformation of a detectable product by physical association. The signalmay be generated by formation of a detectable product by proximityassociation. The signal generated by proximity association may compriseFörster resonance energy transfer (FRET). The proximity association maycomprise association with a complementation enzyme. The signal may begenerated by a single reaction. The signal may be generated by aplurality of reactions. The plurality of reactions may occur in series.The plurality of reactions may occur in parallel. The plurality ofreactions may comprise one or more repetitions of a reaction. Forexample, the reaction may comprise a hybridization reaction or ligationreaction. The reaction may comprise a hybridization reaction and aligation reaction.

The method may further comprise repeating operations 1420, 1430, and1440 one or more times. Different solutions may be directed to theplanar array during rotation of the substrate for consecutive cycles.

Many variations, alterations, and adaptations based on the method 1400provided herein are possible. For example, the order of the operationsof the method 1400 may be changed, some of the operations removed, someof the operations duplicated, and additional operations added asappropriate. Some of the operations may be performed in succession. Someof the operations may be performed in parallel. Some of the operationsmay be performed once. Some of the operations may be performed more thanonce. Some of the operations may comprise sub-operations. Some of theoperations may be automated. Some of the operations may be manual.

FIG. 26 shows a first example of a system 1500 for isolating an analyte.The system may comprise a plurality of linkers 1510 a, 1510 b, 1510 c,and 1510 d. The plurality of linkers may be adhered or otherwise affixedto substrate 310 described herein. For instance, each linker may bebound to a particular individually addressable location of the pluralityof individually addressable locations described herein. Linkers 1510 a,1510 b, 1510 c, and 1510 d may comprise any linker described herein.Some or all of linkers 1510 a, 1510 b, 1510 c, and 1510 d may be thesame. Some or all of linkers 1510 a, 1510 b, 1510 c, and 1510 d may bedifferent. The linkers may be configured to interact with analytes 1520a and 1520 b. For instance, the linkers may be configured to bind toanalytes 1520 a and 1520 b through any interaction described herein.Analytes 1520 a and 1520 b may comprise any analyte described herein.Analytes 1520 a and 1520 b may be the same. Analytes 1520 a and 1520 bmay be different. The linkers may be configured to interact specificallywith particular analytes and/or types thereof. For instance, linker 1510b may be configured to interact specifically with analyte 1520 a. Linker1510 d may be configured to interact specifically with analyte 1520 b.Any linker may be configured to interact with any analyte. In thismanner, specific analytes may be bound to specific locations on thesubstrate. Though shown as comprising four linkers and two analytes inFIG. 26, system 1500 may comprise any number of linkers and analytes.For instance, system 1500 may comprise at least 1, at least 2, at least5, at least 10, at least 20, at least 50, at least 100, at least 200, atleast 500, at least 1,000, at least 2,000, at least 5,000, at least10,000, at least 20,000, at least 50,000, at least 100,000, at least200,000, at least 500,000, at least 1,000,000, at least 2,000,000, atleast 5,000,000, at least 10,000,000, at least 20,000,000, at least50,000,000, at least 100,000,000, at least 200,000,000, at least500,000,000, at least 1,000,000,000 linkers, or a number of linkers thatis within a range defined by any two of the preceding values. System1500 may comprise at least 1, at least 2, at least 5, at least 10, atleast 20, at least 50, at least 100, at least 200, at least 500, atleast 1,000, at least 2,000, at least 5,000, at least 10,000, at least20,000, at least 50,000, at least 100,000, at least 200,000, at least500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, atleast 10,000,000, at least 20,000,000, at least 50,000,000, at least100,000,000, at least 200,000,000, at least 500,000,000, at least1,000,000,000 analytes, or a number of analytes that is within a rangedefined by any two of the preceding values.

FIG. 27 shows a second example of a system 1600 for isolating ananalyte. The system may comprise a well configured to physically trap aparticle. The well may comprise an individually addressable location ofthe plurality of individually addressable locations described herein.The well may be configured to trap an analyte. For instance, the wellmay be configured to trap a droplet of blood 1630. For example, thedroplet of blood may comprise white blood cells 1640, red blood cells1650, and circulating tumor cells 1660. The well may be configured totrap any other analyte described herein. The well may be constructed inlayers using microfabrication materials and techniques. For instance,the well may comprise a base layer 1605. The base layer may comprisesilicon. The well may comprise an oxide layer 1610. The oxide layer maycomprise silicon oxide. The well may comprise a metal layer 1615. Themetal may comprise nickel or aluminum. The well may comprise a nanotubelayer 1620. The nanotube layer may comprise one or more carbonnanotubes. The well may comprise a confinement layer 1625. Theconfinement layer may comprise a photoresist. The photoresist maycomprise SU-8. The nanotube layer and confinement layer may beconfigured to together trap the cell.

FIG. 28 shows examples of control systems to compensate for velocitygradients during scanning. Such control system may algorithmicallycompensate for velocity gradients. The control system may predictive oradaptively compensate for tangential velocity gradients. In a firstcontrol system, illustrated on the left of FIG. 28, the control systemmay, based on scanning of a rotating substrate, measure residual(uncorrected) velocity errors during scanning, compute a compensationcorrection factor, and use the compensation correction factor to set (oradjust) a compensation factor to reduce the velocity errors forsubsequent scanning results. The first control system may be a closedloop control system that removes (or otherwise reduces) velocity errors.In a second control system, illustrated on the right of FIG. 28, thecontrol system may, based on knowledge of the geometry and relativeposition of the scanning relative to the substrate, directly compute (orpredict) the expected velocity gradient, and set (or adjust) the systemto remove the expected gradient.

Multi-Head Imaging Using a Common Linear Motion

Systems and methods described herein may utilize multiple imaging heads(e.g., detector systems, such detector systems comprising a sensor andan illumination source), with each imaging head responsible for imagingdifferent locations on a substrate described herein. For instance, asdescribed herein, a first imaging head may image the substrate along afirst imaging path. The first imaging path may comprise a first seriesof (one or more) rings, a first series of (one or more) spirals, or adifferent first imaging path. Second, third, fourth, fifth, sixth,seventh, eighth, ninth, or tenth imaging heads may image the substratealong second, third, fourth, fifth, sixth, seventh, eighth, ninth, ortenth imaging paths. The second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth imaging paths may comprise second, third,fourth, fifth, sixth, seventh, eighth, ninth, or tenth series of rings,second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenthspirals, or different second, third, fourth, fifth, sixth, seventh,eighth, ninth, or tenth imaging paths. An imaging path or scan path maybe an imaging path or scan path on the substrate or on the sample.

Such multi-head imaging systems and methods may increase a rate ofimaging of the substrate and/or decrease an amount of time that may berequired to image the substrate. In some cases, multiple imaging headsmay move independently relative to the substrate, such as byindependently controlling motions of each of the imaging heads.

As described herein, during detection (e.g., imaging) of a substrate orregion thereof, the substrate may be stationary and one or more detectorsystems or components thereof may be in motion (e.g., rotating). Forexample, the substrate may be stationary and both a sensor (e.g.,line-scan camera) and an illumination source of a detector system may bein motion (e.g., rotating) during detection. Alternatively, thesubstrate may be in motion (e.g., rotating) and one or more detectorsystems or components thereof may be stationary. In some cases, thesubstrate and a detector system or component thereof may be in motion.For example, the substrate may be rotating, and a sensor and anillumination source of a detector system may be in motion. For instance,the sensor and illumination source may translate (e.g., radiallytranslate) across the substrate or the sensor and illumination sourcemay remain disposed in a same physical location but may rotate about acentral axis of the detector system.

The required motions of the imaging heads may be reduced by moving thesubstrate relative to each of the imaging heads such that each of theimaging heads shares a single linear motion with respect to thesubstrate. Such an improvement may be achieved by positioning each scanhead at a different initial distance (e.g., radial distance) from acenter of the substrate and operating each scan head at a different scanrate which depend on the scan head's initial distance from the center ofthe substrate. The single shared linear motion may be along a linearvector. For example, the single shared linear motion may result inradial motion (e.g., directed through an axis of rotation) or non-radialmotion (e.g., not directed through an axis of rotation) of one or morescan heads. In some cases, the imaging heads may be configured to moverelative to the substrate in a radial direction, r, in a polarcoordinate system comprising radial component r and angular component φ.In some cases, the imaging heads may be configured to move in a lineardirection relative to the substrate that is not entirely radial, forexample in a direction comprising both r and φ components. The imagingheads may operate on the same side of the axis of rotation of thesubstrate or on opposite sides of the axis of rotation of the substrate.In the case of non-radial linear motion of the one or more heads, thescan direction of each imaging head may rotate due to a change in anglerelative to the axis of rotation. Such rotations may be compensated bycounter-rotating (for instance, using a prism) to allow for a fixed scandirection for each imaging head.

FIG. 29A shows motion of a substrate relative to two imaging headslocated on the same side of an axis of rotation of the substrate. Thesubstrate 310 may be any substrate described herein. A first imaginghead 1005 may be similar to any first imaging head described herein. Asecond imaging head 1015 may be similar to any second imaging headdescribed herein. At a first moment in time, the first imaging head 1005and second imaging head 1015 may be located on the same side of an axisof rotation 305 of the substrate, such that the first imaging head 1005traces a first imaging path 1010 during rotation of the substrate andthe second imaging head 1015 traces a second imaging path 1020 duringrotation of the substrate. The substrate may be configured to move in alinear, radial direction 1810 relative to the first and second imagingheads. For example, the substrate may be configured to move in a radialdirection, r, in a polar coordinate system comprising radial component rand angular component φ. In some cases, the substrate may be configuredto move in a linear direction that is not entirely radial, for examplein a direction comprising both r and φ components. Thus, the first andsecond imaging paths may vary in location with respect to the substrateover the course of time. Each imaging head may be in opticalcommunication with an imaging field. For example, the first and secondimaging heads may be in optical communication with a first and secondimaging fields, respectively. Each of the first and second imagingfields may be configured to rotate with respect to the substrate, asdescribed elsewhere herein. Rotation of the first and second imagingfields may be independent, or rotation of the first, second, third, orfourth imaging fields may be coordinated.

FIG. 29B shows motion of a substrate relative to two imaging headslocated on opposite sides of an axis of rotation of the substrate. Incomparison with FIG. 29A, at a first moment in time, the first imaginghead 1005 and second imaging head 1015 may be located on opposite sidesof an axis of rotation 305 of the substrate, such that the first imaginghead 1005 traces a first imaging path 1010 during rotation of thesubstrate and the second imaging head 1015 traces a second imaging path1020 during rotation of the substrate. The substrate may be configuredto move in a linear, radial direction 1810 relative to the first andsecond imaging heads. Thus, the first and second imaging paths may varyin location with respect to the substrate over the course of time. Eachimaging head may be in optical communication with an imaging field. Forexample, the first and second imaging heads may be in opticalcommunication with a first and second imaging fields, respectively. Eachof the first and second imaging fields may be configured to rotate withrespect to the substrate, as described elsewhere herein. Rotation of thefirst and second imaging fields may be independent, or rotation of thefirst, second, third, or fourth imaging fields may be coordinated.

FIG. 29C shows motion of a substrate relative to three imaging heads. Athird imaging head 1025 may be similar to any third imaging headdescribed herein. At a first moment in time, the first imaging head 1005may be located on one side of an axis of rotation 305 of the substrate,with respect to a plane containing the axis of rotation, and the secondimaging head 1015 and third imaging head 1025 may be located on theopposite side of the axis of rotation of the substrate, such that thefirst imaging head 1005 traces a first imaging path 1010 during rotationof the substrate, the second imaging head 1015 traces a second imagingpath 1020 during rotation of the substrate, and the third imaging head1025 traces a third imaging path 1030 during rotation of the substrate.The substrate may be configured to move in a linear, radial direction1810 relative to the first, second, and third imaging heads. Thus, thefirst, second, and third imaging paths may vary in location with respectto the substrate over the course of time. Each imaging head may be inoptical communication with an imaging field. For example, the first,second, and third imaging heads may be in optical communication with afirst, second, and third imaging fields, respectively. Each of thefirst, second, and third imaging fields may be configured to rotate withrespect to the substrate, as described elsewhere herein. Rotation of thefirst, second, and third imaging fields may be independent, or rotationof the first, second, and third imaging fields may be coordinated.

FIG. 29D shows motion of a substrate relative to four imaging heads. Afourth imaging head 1035 may be similar to any fourth imaging headdescribed herein. At a first moment in time, the first imaging head 1005and the fourth imaging head 1035 may be located on one side of an axisof rotation 305 of the substrate, with respect to a plane containing theaxis of rotation, and the second imaging head 1015 and third imaginghead 1025 may be located on the opposite side of the axis of rotation ofthe substrate, such that the first imaging head 1005 traces a firstimaging path 1010 during rotation of the substrate, the second imaginghead 1015 traces a second imaging path 1020 during rotation of thesubstrate, the third imaging head 1025 traces a third imaging path 1030,and the fourth imaging head 1025 traces a fourth imaging path 1030during rotation of the substrate. The substrate may be configured tomove in a linear, radial direction 1810 relative to the first, second,third, and fourth imaging heads. Thus, the first, second, third, andfourth imaging paths may vary in location with respect to the substrateover the course of time. Each imaging head may be in opticalcommunication with an imaging field. For example, the first, second,third, and fourth imaging heads may be in optical communication with afirst, second, third, and fourth imaging fields, respectively. Each ofthe first, second, third, and fourth imaging fields may be configured torotate with respect to the substrate, as described elsewhere herein.Rotation of the first, second, third, or fourth imaging fields may beindependent, or rotation of the first, second, third, or fourth imagingfields may be coordinated.

FIG. 29E shows a further embodiment of motion of a substrate relative tofour imaging heads. The first, second, third, and fourth imaging heads,1005, 1015, 1025, and 1035, respectively, may be similar to any imaginghead described herein. At a first moment in time, the first imaging head1005 and the second imaging head 1015 may be located on one side of anaxis of rotation 305 of the substrate, with respect to a planecontaining the axis of rotation, and the third imaging head 1025 and thefourth imaging head 1035 may be located on the opposite side of the axisof rotation of the substrate, such that the first imaging head 1015 andthe fourth imaging head 1035 trace a first half and a second half,respectively, of a first imaging path 1010, and the second imaging head1015 and the third imaging head 1025 trace a first half and a secondhalf, respectively, of a second imaging path 1020 during rotation of thesubstrate 310. The substrate may be configured to move in a linear,radial direction 1810 relative to the first, second, third, and fourthimaging heads. Thus, the first, second, third, and fourth imaging headsmay subsequently trace first and second halves of a third imaging path1030 and a fourth imaging path 1040. The first, second, third, andfourth imaging paths may vary in location with respect to the substrateover the course of time. Each imaging head may be in opticalcommunication with an imaging field. For example, the first, second,third, and fourth imaging heads may be in optical communication with afirst, second, third, and fourth imaging fields, respectively. Each ofthe first, second, third, and fourth imaging fields may be configured torotate with respect to the substrate, as described elsewhere herein.Rotation of the first, second, third, or fourth imaging fields may beindependent, or rotation of the first, second, third, or fourth imagingfields may be coordinated.

FIG. 29F shows a further embodiment of motion of a substrate relative tofour imaging heads. The first, second, third, and fourth imaging heads,1005, 1015, 1025, and 1035, respectively, may be similar to any imaginghead described herein. At a first moment in time, the first imaging head1005 and the second imaging head 1015 may be located on one side of anaxis of rotation 305 of the substrate, with respect to a planecontaining the axis of rotation, and the third imaging head 1025 and thefourth imaging head 1035 may be located on the opposite side of the axisof rotation of the substrate, such that the first imaging head 1005traces a first imaging path 1010, the second imaging head 1015 traces asecond imaging path 1020, the third imaging head 1025 traces a thirdimaging path 1030, and the fourth imaging head 1035 traces a fourthimaging path 1040 during rotation of the substrate. The heads may beconfigured to translate in a linear direction. The translation may beradial, or the translation may not be radial. Translation of one or moreof the first, second, third, or fourth imaging heads may be coupled.Alternatively or in addition, translation of the first, second, third,or fourth imaging heads may be independent. In some embodiments,translation of the first and second imaging heads may be coupled, andtranslation of the third and fourth imaging heads may be coupled. Thus,the first, second, third, and fourth imaging paths may vary in locationwith respect to the substrate over the course of time. Each imaging headmay be in optical communication with an imaging field. For example, thefirst, second, third, and fourth imaging heads may be in opticalcommunication with a first, second, third, and fourth imaging fields,respectively. Each of the first, second, third, and fourth imagingfields may be configured to rotate with respect to the substrate, asdescribed elsewhere herein. Rotation of the first, second, third, orfourth imaging fields may be independent, or rotation of the first,second, third, or fourth imaging fields may be coordinated.

FIG. 29G shows a further embodiment of motion of a substrate relative tofour imaging heads. The first, second, third, and fourth imaging heads,1005, 1015, 1025, and 1035, respectively, may be similar to any imaginghead described herein. At a first moment in time, the first imaging head1005, the second imaging head 1015, the third imaging head 1025, and thefourth imaging head 1035 may be located on the same side of an axis ofrotation 305 of the substrate, with respect to a plane containing theaxis of rotation. The first imaging head 1005 may trace a first imagingpath 1010, the second imaging head 1015 may trace a second imaging path1020, the third imaging head 1025 may trace a third imaging path 1030,and the fourth imaging head 1035 may trace a fourth imaging path 1040during rotation of the substrate. The heads may be configured totranslate in a linear direction. The translation may be radial, or thetranslation may not be radial. Translation of the first, second, third,or fourth imaging heads may be independent. Thus, the first, second,third, and fourth imaging paths may vary in location with respect to thesubstrate over the course of time. Each imaging head may be in opticalcommunication with an imaging field. For example, the first, second,third, and fourth imaging heads may be in optical communication with afirst, second, third, and fourth imaging fields, respectively. Each ofthe first, second, third, and fourth imaging fields may be configured torotate with respect to the substrate, as described elsewhere herein.Rotation of the first, second, third, or fourth imaging fields may beindependent, or rotation of the first, second, third, or fourth imagingfields may be coordinated.

FIG. 30A shows successive ring paths of two imaging heads located on thesame side of an axis of rotation of a substrate. At a first moment intime, the first imaging head (not depicted in FIG. 30A) and secondimaging head (not depicted in FIG. 30A) may be located on the same sideof an axis of rotation 305 of the substrate 310, such that the firstimaging head traces a first imaging path 1010 a at a first time pointduring rotation of the substrate and the second imaging head traces asecond imaging path 1020 a at the first time point during rotation ofthe substrate. For example, the two imaging heads may be located andconfigured as in FIG. 29A. As the substrate moves in a linear, radialdirection 1810 relative to the first and second imaging heads, the firstand second imaging heads may trace a series of imaging paths duringrotation of the substrate. For instance, if the first and second imagingheads are located on the same side of the axis of rotation of thesubstrate, the first imaging head may trace imaging path 1010 b at asecond time point, imaging path 1010 c at a third time point, andimaging path 1010 d at a fourth time point while the second imaging pathmay trace imaging path 1020 b at the second time point, imaging path1020 c at the third time point, and imaging path 1020 d at the fourthtime point. When the first and second imaging heads are located on thesame side of the axis of rotation, the succession of imaging paths {1010a, 1010 b, 1010 c, 1010 d} and {1020 a, 1020 b, 1020 c, 1020 d} mayproceed in the same direction with respect to the substrate. Forinstance, as depicted in FIG. 30A, the succession of imaging paths {1010a, 1010 b, 1010 c, 1010 d} and {1020 a, 1020 b, 1020 c, 1020 d} may bothproceed in a direction toward the center of the substrate.

FIG. 30B shows successive ring paths of two imaging heads located onopposite sides of an axis of rotation of a substrate. In comparison withFIG. 30A, at a first moment in time, the first imaging head (notdepicted in FIG. 30B) and second imaging head (not depicted in FIG. 30B)may be located on opposite sides of an axis of rotation 305 of thesubstrate, such that the first imaging head traces a first imaging path1010 a at a first time point during rotation of the substrate and thesecond imaging head traces a second imaging path 1020 a at the firsttime point during rotation of the substrate. For example, the twoimaging heads may be located and configured as in FIG. 29B. As thesubstrate moves in a linear, radial direction 1810 relative to the firstand second imaging heads, one of the heads moves towards the centralaxis and the other head moves away from the central axis, the first andsecond imaging heads each tracing a series of imaging paths duringrotation of the substrate. For instance, if the first and second imagingheads are located on opposite sides of the axis of rotation of thesubstrate, the first imaging head may trace imaging path 1010 b at asecond time point, imaging path 1010 c at a third time point, andimaging path 1010 d at a fourth time point while the second imaging pathmay trace imaging path 1020 b at the second time point, imaging path1020 c at the third time point, and imaging path 1020 d at the fourthtime point. When the first and second imaging heads are located on theopposite sides of the axis of rotation, the succession of imaging paths{1010 a, 1010 b, 1010 c, 1010 d} and {1020 a, 1020 b, 1020 c, 1020 d}may proceed in opposite directions with respect to the substrate. Forinstance, as depicted in FIG. 30B, the succession of imaging paths {1010a, 1010 b, 1010 c, 1010 d} may proceed in a direction toward the centerof the substrate while the succession of imaging paths {1020 a, 1020 b,1020 c, 1020 d} may proceed in a direction away from the center of thesubstrate.

FIG. 30C shows staggered ring paths of two imaging heads located on thesame side of an axis of rotation of a substrate. At a first moment intime, the first imaging head (not depicted in FIG. 30C) and secondimaging head (not depicted in FIG. 30C) may be located on the same sideof an axis of rotation 305 of the substrate 310, such that the firstimaging head traces a first imaging path 1010 a at a first time pointduring rotation of the substrate and the second imaging head traces asecond imaging path 1020 a at the first time point during rotation ofthe substrate. As the substrate moves in a linear, radial direction 1810relative to the first and second imaging heads, the first and secondimaging heads may trace a series of imaging paths during rotation of thesubstrate. For instance, if the first and second imaging heads arelocated on the same side of the axis of rotation of the substrate, thefirst imaging head may trace imaging path 1010 b at a second time point,imaging path 1010 c at a third time point, and imaging path 1010 d at afourth time point while the second imaging path may trace imaging path1020 b at the second time point, imaging path 1020 c at the third timepoint, and imaging path 1020 d at the fourth time point. The successionof imaging paths {1010 a, 1010 b, 1010 c, 1010 d} and {1020 a, 1020 b,1020 c, 1020 d} may be staggered, such that successive imaging pathstoward or away from the center of the substrate are traced byalternating imaging heads. When the first and second imaging heads arelocated on the same side of the axis of rotation, the succession ofimaging paths {1010 a, 1010 b, 1010 c, 1010 d} and {1020 a, 1020 b, 1020c, 1020 d} may proceed in the same direction with respect to thesubstrate. For instance, as depicted in FIG. 30C, the succession ofimaging paths {1010 a, 1010 b, 1010 c, 1010 d} and {1020 a, 1020 b, 1020c, 1020 d} may both proceed in a direction toward the center of thesubstrate.

FIG. 30D shows staggered ring paths of two imaging heads located onopposite sides of an axis of rotation of a substrate. At a first momentin time, the first imaging head (not depicted in FIG. 30D) and secondimaging head (not depicted in FIG. 30D) may be located on opposite sidesof an axis of rotation 305 of the substrate 310, such that the firstimaging head traces a first imaging path 1010 a at a first time pointduring rotation of the substrate and the second imaging head traces asecond imaging path 1020 a at the first time point during rotation ofthe substrate. As the substrate moves in a linear, radial direction 1810relative to the first and second imaging heads, one of the heads movestowards the central axis and the other head moves away from the centralaxis, the first and second imaging heads each tracing a series ofimaging paths during rotation of the substrate. For instance, if thefirst and second imaging heads are located on opposite sides of the axisof rotation of the substrate, the first imaging head may trace imagingpath 1010 b at a second time point, imaging path 1010 c at a third timepoint, and imaging path 1010 d at a fourth time point while the secondimaging path may trace imaging path 1020 b at the second time point,imaging path 1020 c at the third time point, and imaging path 1020 d atthe fourth time point. The succession of imaging paths {1010 a, 1010 b,1010 c, 1010 d} and {1020 a, 1020 b, 1020 c, 1020 d} may be staggered,such that successive imaging paths toward or away from the center of thesubstrate are traced by alternating imaging heads. When the first andsecond imaging heads are located on the opposite sides of the axis ofrotation, the succession of imaging paths {1010 a, 1010 b, 1010 c, 1010d} and {1020 a, 1020 b, 1020 c, 1020 d} may proceed in oppositedirections with respect to the substrate. For instance, as depicted inFIG. 30D, the succession of imaging paths {1010 a, 1010 b, 1010 c, 1010d} may proceed in a direction toward the center of the substrate whilethe succession of imaging paths {1020 a, 1020 b, 1020 c, 1020 d} mayproceed in a direction away from the center of the substrate.

FIG. 31A-FIG. 31B show rotating scan directions of an imaging head dueto non-radial motion of the head relative to a substrate (e.g., motioncomprising both r and φ components in a polar coordinate system). Forexample, as shown in FIG. 31A, the head may be moving along direction316 relative to the substrate, which is not through the central axis. Ata first point in time, the first imaging head (not depicted in FIG. 31A)or second imaging head (not depicted in FIG. 31A) may be locatedoff-axis from a longitudinal axis 315 of the substrate 310. In such acase, the first or second imaging head may have a tangential velocityrelative to the substrate that changes in direction as the substratemoves with respect to the first or second imaging head. For instance, asdepicted in FIG. 31A, the second imaging head may have a tangentialvelocity vector 2020 a relative to the substrate while tracing theimaging path 1020 a and a tangential velocity vector 2020 b relative tothe substrate while tracing the imaging path 1020 c. As shown in FIG.31, the tangential velocity vectors 2020 a and 2020 b may point insubstantially different directions. Such an effect may be manifested asa rotation of the imaging field as the first imaging head traces thesuccession of imaging paths {1010 a, 1010 b, 1010 c, 1010 d} or as thesecond imaging head traces the succession of imaging paths {1020 a, 1020b, 1020 c, 1020 d}.

FIG. 31B shows rotating scan directions of imaging fields of view due tonon-radial motion of the imaging head relative to the substrate. Forexample, a first imaging head (not shown in FIG. 31B) imaging a firstfield of view 3101, and a third imaging head (not shown in FIG. 31B)imaging a third field of view 3103 may translate relative to thesubstrate 310 in directions 3111 and 3113, respectively, that are notthrough the central axis. At a first point in time, the first imagingfield 3101 or the third imaging field 3103 may be located off-axis froma longitudinal axis 315 of the substrate 310. In such a case, the firstor third imaging field may have a tangential velocity relative to thesubstrate that changes in direction as the substrate moves with respectto the first or second imaging head. Following non-radial translation,the first and third imaging fields may no longer be positionedperpendicular to the tangential motion of the substrate (indicated bygray rectangles). In some embodiments the first and third imaging fieldsmay undergo a counter-rotation with respect to the substrate followingnon-radial translation such that the first and third imaging fields maybe positioned perpendicular to the tangential motion of the substrate(indicated by dashed rectangles). Counter-rotation may be achieved usingany of the methods disclosed herein, such as those described withrespect to FIG. 34A-FIG. 34C.

FIG. 34A-FIG. 34C shown exemplary optical systems for rotating animaging field. Such a rotation of the imaging field may be compensatedby counter-rotating the imaging field. For instance, the imaging fieldmay be counter-rotated using a prism system, such as a delta rotatorprism, a Schmidt rotator, or a Dove prism. An exemplary optical systemfor counter-rotating an imaging field using a Dove prism is shown inFIG. 34B. Alternatively or in addition, the compensation may be achievedby using one or more mirrors or other optical elements (e.g.,beamsplitter (e.g., dichroic mirror)), as described herein.Alternatively or in addition, the compensation may be achieved byrotating one or more sensors in the optical head(s). For example, thecompensation may be achieved by rotating a detector (e.g., a line-scancamera) and a line shaping element (e.g., a cylindrical lens). Exemplaryoptical systems for rotating a detector and a line shaping element areshown in FIG. 34A and FIG. 34C. The imaging field may be rotated aboutan axis of rotation, which may be counter to the axis of rotation of thesurface, to compensate for a relative translational motion that may notintersect the axis of rotation of the surface and the imaging field, asshown in FIG. 33.

FIG. 35A-FIG. 35C illustrate exemplary optical path trajectories of anoptical system 3500 comprising imaging heads. Two imaging heads 3501 and3502, each comprising an objective, may be positioned to imagecorresponding regions of a substrate 3503, as shown in FIG. 35A. Theimaging heads may be positioned on opposite sides of a radial line 3504.In some embodiments, the two imaging heads may be positioned atdifferent distances from the radial line. The distances from the radialline may be determined by the diameter of the objectives and opticalpath trajectories of the imaging heads. The substrate may be configuredto rotate about an axis of rotation 3505 and translate along an axis oftranslation 3506 with respect to the imaging heads. The substrate may berotated about the axis of rotation such that the two imaging heads tracecircular optical path trajectories. Ideal optical path trajectories inwhich an entire outer region of the surface is scanned without overlapare outlined with solid lines in FIG. 35A-FIG. 35C. An optical pathtrajectory resulting from coordinated motion of two imaging heads inwhich the optical path trajectories partially overlap is outlined withdashes in FIG. 35A-FIG. 35C. For clarity, only the initial position ofthe imaging heads 3501 and 3502 are shown in FIG. 35A-FIG. 35C.

The first optical path 3511 of the first imaging head 3501 may beconcentric to the first optical path 3521 of the second imaging head3502, as shown in FIG. 35C. Upon translation of the substrate along theaxis of translation, the first and second imaging heads may move tosecond optical paths 3512 and 3522, third optical paths 3513 and 3523,third optical paths, fourth optical paths 3514 and 3524, fifth opticalpaths 3515 and 3525, sixth optical paths 3516 and 3526, seventh opticalpaths 3517 and 3527, or more optical paths. In some embodiments, theoptical path trajectories of the two imaging heads partially overlap,with the amount of overlap increasing for optical paths closer to theaxis of rotation of the substrate. The optical path trajectories and thedistances of the imaging heads from the radial line may be optimized forminimal overlap of the optical path trajectories of the two imagingheads, as shown in FIG. 35B. The optical path trajectories may overlapby no more than 0.10%, no more than 0.20%, no more than 0.50%, no morethan 1%, no more than 2%, no more than 3%, no more than 4%, no more than5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%,no more than 10%, no more than 15%, no more than 20%, no more than 30%,no more than 40%, or no more than 50%.

In some embodiments, the optical path trajectories of the two imagingheads do not substantially overlap. In some embodiments, the opticalpath trajectories of the two imaging heads are partially separated, withthe amount of separation decreasing for optical paths closer to the axisof rotation of the substrate. The optical path trajectories and thedistances of the imaging heads from the radial line may be optimized toreduce the amount of substrate that is not scanned without substantialoverlap of the optical path trajectories of the two imaging heads (notshown in FIG. 32). In some instances, the unscanned portion of thesubstrate may comprise no more than 0.10%, no more than 0.20%, no morethan 0.50%, no more than 1%, no more than 2%, no more than 3%, no morethan 4%, no more than 5%, no more than 6%, no more than 7%, no more than8%, no more than 9%, no more than 10%, no more than 15%, no more than20%, no more than 30%, no more than 40%, or no more than 50% of thetotal substrate surface. In some cases, the optical path trajectories ofthe tow imaging heads may be configured to reduce the amount of overlapin order to reduce the amount of photodamage to the substrate or areagent on the substrate.

The substrate motions described herein, for example those described withrespect to FIG. 29-FIG. 31, may be used to scan a surface comprising ananalyte. In some cases, scanning the surface may comprise detecting theanalyte on the surface. FIG. 32 shows a flowchart for an example of amethod 2100 for analyte detection or analysis. In a first operation2110, the method 2100 may comprise rotating an open substrate about acentral axis, the open substrate having an array of immobilized analytesthereon.

In a second operation 2120, the method 2100 may comprise delivering asolution having a plurality of probes to a region proximal to thecentral axis to introduce the solution to the open substrate.

In a third operation 2130, the method 2100 may comprise dispersing thesolution across the open substrate (for instance, at least bycentrifugal force) such that at least one of the plurality of probesbinds to at least one of the immobilized analytes to form a bound probe.

In a fourth operation 2140, the method 2100 may comprise, duringrotation of the open substrate, simultaneously using a first detector toperform a first scan of the open substrate along a first set of one ormore scan paths and a second detector to perform a second scan of theopen substrate along a second set of one or more scan paths. The firstset of one or more scan paths and the second set of one or more scanpaths may be different. The first detector or the second detector maydetect at least one signal from the bound probe. The first detector maybe disposed at a first radial position relative to the central axis. Thesecond detector is disposed at a second radial position relative to thecentral axis. The first detector and the second detector may undergorelative motion with respect to the central axis along a same linearvector, to generate the first set of one or more scan paths and thesecond set of one or more scan paths, respectively.

The first detector and the second detector may operate at different scanrates. For instance, the different scan rates of the first detector andthe second detector may be a function of the first radial position andthe second radial position, respectively. Alternatively, the detectorsmay operate at a fixed line rate. For example, algorithmic processingmay resolve oversampling of the optical head located in the inner radialpositions.

The first set of one or more scan paths may comprise one or morecircular scan paths having different radii. For instance, the first setof one or more scan paths may comprise at least about 1, at least about2, at least about 3, at least about 4, at least about 5, at least about6, at least about 7, at least about 8, at least about 9, at least about10, at least about 20, at least about 30, at least about 40, at leastabout 50, at least about 60, at least about 70, at least about 80, atleast about 90, at least about 100, or more circular scan paths, at mostabout 100, at most about 90, at most about 80, at most about 70, at mostabout 60, at most about 50, at most about 40, at most about 30, at mostabout 20, at most about 10, at most about 9, at most about 8, at mostabout 7, at most about 6, at most about 5, at most about 4, at mostabout 3, at most about 2, or at most about 1 circular scan paths, or anumber of circular scan paths that is within a range defined by any twoof the preceding values.

The second set of one or more scan paths may comprise one or morecircular scan paths having different radii. For instance, the second setof one or more scan paths may comprise at least about 1, at least about2, at least about 3, at least about 4, at least about 5, at least about6, at least about 7, at least about 8, at least about 9, at least about10, at least about 20, at least about 30, at least about 40, at leastabout 50, at least about 60, at least about 70, at least about 80, atleast about 90, at least about 100, or more circular scan paths, at mostabout 100, at most about 90, at most about 80, at most about 70, at mostabout 60, at most about 50, at most about 40, at most about 30, at mostabout 20, at most about 10, at most about 9, at most about 8, at mostabout 7, at most about 6, at most about 5, at most about 4, at mostabout 3, at most about 2, or at most about 1 circular scan paths, or anumber of circular scan paths that is within a range defined by any twoof the preceding values.

The first set of one or more scan paths may comprise one or more spiralscan paths. For instance, the first set of one or more scan paths maycomprise at least about 1, at least about 2, at least about 3, at leastabout 4, at least about 5, at least about 6, at least about 7, at leastabout 8, at least about 9, at least about 10, at least about 20, atleast about 30, at least about 40, at least about 50, at least about 60,at least about 70, at least about 80, at least about 90, at least about100, or more spiral scan paths, at most about 100, at most about 90, atmost about 80, at most about 70, at most about 60, at most about 50, atmost about 40, at most about 30, at most about 20, at most about 10, atmost about 9, at most about 8, at most about 7, at most about 6, at mostabout 5, at most about 4, at most about 3, at most about 2, or at mostabout 1 spiral scan paths, or a number of spiral scan paths that iswithin a range defined by any two of the preceding values.

The second set of one or more scan paths may comprise one or more spiralscan paths. For instance, the second set of one or more scan paths maycomprise at least about 1, at least about 2, at least about 3, at leastabout 4, at least about 5, at least about 6, at least about 7, at leastabout 8, at least about 9, at least about 10, at least about 20, atleast about 30, at least about 40, at least about 50, at least about 60,at least about 70, at least about 80, at least about 90, at least about100, or more spiral scan paths, at most about 100, at most about 90, atmost about 80, at most about 70, at most about 60, at most about 50, atmost about 40, at most about 30, at most about 20, at most about 10, atmost about 9, at most about 8, at most about 7, at most about 6, at mostabout 5, at most about 4, at most about 3, at most about 2, or at mostabout 1 spiral scan paths, or a number of spiral scan paths that iswithin a range defined by any two of the preceding values.

The same linear vector may be in a radial direction through the centralaxis. The same linear vector may not be in a radial direction (e.g., notthrough the central axis). The method may further comprise compensatingfor velocity differences (such as tangential velocity differences, asdescribed herein with respect to FIG. 31) of different areas atdifferent radial positions with respect to the central axis. A givenscan path of the first set of one or more scan paths may comprise thedifferent areas. A given scan path of the second set of one or more scanpaths may comprise the different areas. The compensating may compriseusing one or more prisms, such as one or more delta rotator prisms,Schmidt rotators, or Dove prisms.

The first detector and the second detector may be substantiallystationary during the relative motion. The open substrate may undergoboth rotational and translation motion during the relative motion. Thefirst detector and the second detector may undergo motion during therelative motion. The open substrate may undergo rotational motionrelative to the first detector and the second detector and the firstdetector and second detector may undergo linear motion relative to thecentral axis. The first detector may undergo the relative motion duringrotation of the open substrate. The second detector may undergo therelative motion during rotation of the open substrate. The firstdetector may undergo the relative motion when the open substrate issubstantially stationary. The second detector may undergo the relativemotion when the open substrate is substantially stationary.

A given scan path of the first set of one or more scan paths may includean area scanned during the relative motion. A given scan path of thesecond set of one or more scan paths may include an area scanned duringthe relative motion. A given scan path of the first set of one or morescan paths may not include an area scanned during the relative motion. Agiven scan path of the second set of one or more scan paths may notinclude an area scanned during the relative motion.

The first detector and the second detector may have the same angularposition relative to the central axis. The first detector and the seconddetector may have different angular positions relative to the centralaxis. The first detector and second detector may have opposite angularpositions (e.g., having 180 degrees separation) relative to the centralaxis.

The first detector may have an angular position of at least about 1degree, at least about 2 degrees, at least about 3 degrees, at leastabout 4 degrees, at least about 5 degrees, at least about 6 degrees, atleast about 7 degrees, at least about 8 degrees, at least about 9degrees, at least about 10 degrees, at least about 15 degrees, at leastabout 20 degrees, at least about 25 degrees, at least about 30 degrees,at least about 35 degrees, at least about 40 degrees, at least about 45degrees, at least about 50 degrees, at least about 55 degrees, at leastabout 60 degrees, at least about 65 degrees, at least about 70 degrees,at least about 75 degrees, at least about 80 degrees, at least about 81degrees, at least about 82 degrees, at least about 83 degrees, at leastabout 84 degrees, at least about 85 degrees, at least about 86 degrees,at least about 87 degrees, at least about 88 degrees, at least about 89degrees, or more relative to the central axis, at most about 89 degrees,at most about 88 degrees, at most about 87 degrees, at most about 86degrees, at most about 85 degrees, at most about 84 degrees, at mostabout 83 degrees, at most about 82 degrees, at most about 81 degrees, atmost about 80 degrees, at most about 75 degrees, at most about 70degrees, at most about 65 degrees, at most about 60 degrees, at mostabout 55 degrees, at most about 50 degrees, at most about 45 degrees, atmost about 40 degrees, at most about 35 degrees, at most about 30degrees, at most about 25 degrees, at most about 20 degrees, at mostabout 15 degrees, at most about 10 degrees, at most about 9 degrees, atmost about 8 degrees, at most about 7 degrees, at most about 6 degrees,at most about 5 degrees, at most about 4 degrees, at most about 3degrees, at most about 2 degrees, at most about 1 degree, or lessrelative to the central axis, or an angular position relative to thecentral axis that is within a range defined by any two of the precedingvalues.

The second detector may have an angular position of at least about 1degree, at least about 2 degrees, at least about 3 degrees, at leastabout 4 degrees, at least about 5 degrees, at least about 6 degrees, atleast about 7 degrees, at least about 8 degrees, at least about 9degrees, at least about 10 degrees, at least about 15 degrees, at leastabout 20 degrees, at least about 25 degrees, at least about 30 degrees,at least about 35 degrees, at least about 40 degrees, at least about 45degrees, at least about 50 degrees, at least about 55 degrees, at leastabout 60 degrees, at least about 65 degrees, at least about 70 degrees,at least about 75 degrees, at least about 80 degrees, at least about 81degrees, at least about 82 degrees, at least about 83 degrees, at leastabout 84 degrees, at least about 85 degrees, at least about 86 degrees,at least about 87 degrees, at least about 88 degrees, at least about 89degrees, or more relative to the central axis, at most about 89 degrees,at most about 88 degrees, at most about 87 degrees, at most about 86degrees, at most about 85 degrees, at most about 84 degrees, at mostabout 83 degrees, at most about 82 degrees, at most about 81 degrees, atmost about 80 degrees, at most about 75 degrees, at most about 70degrees, at most about 65 degrees, at most about 60 degrees, at mostabout 55 degrees, at most about 50 degrees, at most about 45 degrees, atmost about 40 degrees, at most about 35 degrees, at most about 30degrees, at most about 25 degrees, at most about 20 degrees, at mostabout 15 degrees, at most about 10 degrees, at most about 9 degrees, atmost about 8 degrees, at most about 7 degrees, at most about 6 degrees,at most about 5 degrees, at most about 4 degrees, at most about 3degrees, at most about 2 degrees, at most about 1 degree, or lessrelative to the central axis, or an angular position relative to thecentral axis that is within a range defined by any two of the precedingvalues.

A given scan path of the first set of one or more scan paths may includea first area and a second area. The first area and second area may be atdifferent radial positions of the open substrate with respect to thecentral axis. The first area and second area may be spatially resolvedby the first detector. A given scan path of the second set of one ormore scan paths may include a first area and a second area. The firstarea and second area may be at different radial positions of the opensubstrate with respect to the central axis. The first area and secondarea may be spatially resolved by the second detector.

Reel-to-Reel Processing of Biological Analytes

In some instances, an open substrate system of the present disclosuremay comprise a substantially flexible substrate. For example, thesubstantially flexible substrate may comprise a film. The substantiallyflexible substrate may have any degree of deformability. In someinstances, an open substrate system of the present disclosure mayachieve dispensing via contact with a reagent reservoir or bath. In someinstances, a substantially flexible substrate may be used with a reagentreservoir or bath. In some instances, a substantially rigid substratemay be used with a reagent reservoir or bath. In some instances, asubstantially flexible substrate may be used with other dispensingmechanisms (e.g., nozzles) described herein. In some instances, asubstantially rigid substrate may be used with other dispensingmechanisms (e.g., nozzles) described herein.

In an aspect, provided herein are methods for processing a biologicalanalyte, comprising (a) providing a flexible substrate comprising anarray having immobilized thereto the biological analyte, wherein theflexible substrate is able to be moved through a reel; (b) bringing theflexible substrate in contact with a reservoir comprising a solutionthat comprises a plurality of probes; (c) subjecting the biologicalanalyte to conditions sufficient to conduct a reaction between at leastone probe of the plurality of probes and the biological analyte, tocouple the at least one probe to the biological analyte; and (d)detecting one or more signals from the at least one probe coupled to thebiological analyte, thereby analyzing the biological analyte.

In some embodiments, the method further comprises using a recirculationtank.

In some cases, a dimension of the flexible substrate is the width of afield of view of the imaging method.

In some embodiments, the process of bringing the flexible substrate incontact with a reservoir and/or the process of subjecting the biologicalanalyte to conditions sufficient to conduct a reaction is performedwhile the flexible substrate is moved through the reel.

In some embodiments, the flexible substrate is moved through a reel tocontact the solution with the biological analyte. In some embodiments,the flexible substrate is further moved through a second reel to bringthe flexible substrate in contact with a second reservoir comprising asecond solution. In some cases, the second solution comprises a washbuffer. In some cases, the second solution comprises a plurality ofprobes, wherein the solution and the second solution are different.

In some embodiments, the processes of bringing the flexible substrate incontact with the reservoir, subjecting the biological analyte toconditions sufficient to conduct the reaction, and detecting may berepeated any number of times, for example, a number of times sufficientto complete an assay (e.g., determining a sequence of a nucleic acidmolecule).

In some embodiments, the method further comprises repeating theprocesses of bringing the flexible substrate in contact with thereservoir, subjecting the biological analyte to conditions sufficient toconduct the reaction, and detecting with an additional plurality ofprobes that is different than the plurality of probes. In some cases,the plurality of probes can comprise any probe described elsewhereherein. For example, the probe may comprise an oligonucleotide moleculehaving any length. For example, the probe may comprise oligonucleotides1 to 10 bases in length. A given probe may be a dibase probe. A givenprobe may be between 10 to 20 bases in length. In some instances, theplurality of probes may be labeled.

In some embodiments, the biological analyte is a nucleic acid molecule,and analyzing the biological analyte comprises identifying a sequence ofthe nucleic acid molecule. In some embodiments, the plurality of probesis a plurality of nucleotides. In some embodiments, the plurality ofprobes is a plurality of oligonucleotide molecules. In some cases,subjecting the biological analyte to the conditions sufficient toconduct the reaction comprises subjecting the nucleic acid molecule to aprimer extension reaction under conditions sufficient to incorporate atleast one nucleotide from the plurality of nucleotides into a growingstrand that is complementary to the nucleic acid molecule. In someembodiments, the one or more signals are indicative of incorporation ofat least one nucleotide. In some embodiments, the plurality ofnucleotides comprises nucleotide analogs. In some embodiments, themethod further comprises repeating the processes of bringing theflexible substrate in contact with a reservoir and subjecting thebiological analyte to conditions sufficient to conduct a reaction withan additional plurality of nucleotides that are of a second canonicalbase type, wherein the second canonical base type is different than thefirst canonical base type. In some embodiments, the plurality of probesis a plurality of oligonucleotide molecules. In some embodiments, thebiological analyte is a nucleic acid molecule, and the subjectingcomprises conducting a complementarity binding reaction between the atleast one probe and the nucleic acid molecule to identify a presence ofhomology between the at least one probe and the biological analyte inthe detection.

In some embodiments, the detecting is conducted using a sensor thatcontinuously scans the array. In some embodiments, the detecting isconducted using a sensor that scans the array linearly. In some cases,the detecting is conducted using any sensor or sensing mechanismdescribed herein.

In some embodiments, the method further comprises using a pullingmechanism to move the flexible substrate through the reel and intocontact with the reservoir, thereby dispensing the solution on theflexible substrate. Any other motion units or mechanisms may be used toactuate the flexible substrate.

In some embodiments, the fluid viscosity of the solution or a velocityof the flexible substrate is selected to yield a predetermined thicknessof a layer of the solution adjacent to the array. In some embodiments asqueegee near the substrate may be used to yield a predeterminedthickness of a layer. In some embodiments, the flexible substrate istextured or patterned. In some embodiments the flexible substrate issubstantially planar.

In some embodiments, the flexible substrate comprises an array whichcomprises a plurality of individually addressable locations, and whereinthe biological analyte is disposed at a given individually addressablelocation of the plurality of individually addressable locations. In someembodiments, the array has immobilized thereto one or more additionalbiological analytes.

In some embodiments, bringing the flexible substrate in contact with thereservoir comprises achieving contact at an area of contact between theflexible substrate and the reservoir. In some embodiments, bringing theflexible substrate in contact with the reservoir comprises achievingcontact along a line of contact between the substrate and the reservoir.

In some cases, the biological analyte can comprise any analyte describedelsewhere herein. The analyte may be a single cell analyte. The analytemay be a nucleic acid molecule or clonal population of nucleic acids.The analyte may be a protein molecule. The analyte may be a single cell.The analyte may be a particle. The analyte may be an organism. Theanalyte may be part of a colony. The analyte may be immobilized in anindividually addressable location on the planar array. The array on theflexible substrate may comprise two or more types of analytes. The twoor more types of analytes may be arranged randomly. The two or moretypes of analytes may be arranged in a regular pattern.

In some instances, the analyte can be immobilized to the flexiblesubstrate via a linker. The flexible substrate may comprise the linkerthat is coupled to the analyte. The linker can be any linker describedherein. The linker may comprise a carbohydrate molecule. The linker maycomprise an affinity binding protein. The linker may be hydrophilic. Thelinker may be hydrophobic. The linker may be electrostatic. The linkermay be labeled. The linker may be integral to the substrate. The linkermay be an independent layer on the substrate. In some embodiments, thebiological analyte is coupled to a bead, which bead is immobilized tothe flexible substrate. The method may further comprise, prior toproviding the flexible substrate, directing the biological analyteacross the flexible substrate comprising the linker. The biologicalanalyte may be coupled to a bead, which bead is immobilized to thesubstrate. In some instances, for example, the flexible substratecomprising the linker may be brought into contact with a reservoircomprising a solution comprising the biological analyte. Alternativelyor in addition, the biological analyte may be dispensed onto theflexible substrate in accordance with any other dispensing mechanismdescribed herein.

The method may further comprise recycling a subset of the solution thathas contacted the substrate. The recycling may comprise collecting,filtering, and reusing the subset of the solution. The filtering may bemolecular filtering. For example, the solution in the reservoir (afterthe substrate has passed through) may be recycled.

The signal may be an optical signal. The signal may be a fluorescencesignal. The signal may be a light absorption signal. The signal may be alight scattering signal. The signal may be a luminescent signal. Thesignal may be a phosphorescence signal. The signal may be an electricalsignal. The signal may be an acoustic signal. The signal may be amagnetic signal. The signal may be generated by binding of a label tothe analyte. The label may be bound to a molecule, particle, cell, ororganism. The label may be bound to the analyte (e.g., molecule,particle, cell, or organism) prior to deposition on the substrate. Thelabel may be bound to the analyte subsequent to deposition on thesubstrate. The signal may be generated by formation of a detectableproduct by a chemical reaction. The reaction may comprise an enzymaticreaction. The signal may be generated by formation of a detectableproduct by physical association. The signal may be generated byformation of a detectable product by proximity association. The signalgenerated by proximity association may comprise Förster resonance energytransfer (FRET). The proximity association may comprise association witha complementation enzyme. The signal may be generated by a singlereaction. The signal may be generated by a plurality of reactions. Theplurality of reactions may occur in series. The plurality of reactionsmay occur in parallel. The plurality of reactions may comprise one ormore repetitions of a reaction. The reaction may comprise ahybridization reaction or ligation reaction. The reaction may comprise ahybridization reaction and a ligation reaction.

One or more processes of the methods described herein may be repeated ina continuous fashion. One or more methods described herein may offerhigher efficiency in reagent usage. One or more methods described hereinmay allow for detection of one or more signals at multiple locationsalong the array contemporaneously. In some cases, throughput may bealtered by changing the dimensions of the flexible substrate. Forexample, the flexible substrate may be a rectangular film, wherein awider film allows for increased throughput. In another example, thelength of the reel may be changed to match the detection method.

FIG. 36A-FIG. 36B schematically illustrate methods for processing abiological analyte, as shown in FIG. 36A and FIG. 36B. A flexiblesubstrate such as a film 2710 has immobilized thereto the biologicalanalyte. In some cases, the biological analyte is immobilized to thefilm in an arrayed pattern in individually addressable locations. Inother embodiments, the biological analyte is immobilized to the film ina random orientation. The film 2710 comprising the biological analyteimmobilized thereto is capable of being moved through a reel or a seriesof reels. In process 2712, the film 2710 comprising the biologicalanalyte immobilized thereto is moved through a reel and brought intocontact with a reservoir 2730 comprising a plurality of probes, such asa plurality of labeled probes. In some cases, the labeled probe is afluorescently labeled nucleotide. The labeled probes may couple to asubset of the individually addressable locations comprising thebiological analyte, e.g., based on sequence complementarity. In process2714, the film is then moved through a second reel and brought intocontact with a reservoir 2740 comprising a wash buffer. The wash buffermay allow for removal of uncoupled probes, such as probes that areunbound or unhybridized to the film. Detection of one or more signalsfrom the at least one probe coupled to the biological analyte may beperformed. In process 2715, detection can occur using a sensor, such asan imager 2750, in which an image of the film is taken. In some cases,the field of view of the image is one of the dimensions (e.g., thewidth) of the film. In some cases, detection may occur a plurality oftimes during the processing. For example, as shown in FIG. 36A,detection may occur after one or more wash step following treatment witha probe (e.g., dATP, dCTP, dTTP, dGTP, or dUTP). In some cases, asurface may me imaged prior to treatment with a probe, as shown in FIG.36B. In process 2716, the film 2710 is moved through a third reel andbrought into contact with a reservoir 2760 comprising a plurality ofprobes, such as a plurality of labeled probes. The labeled probes inreservoir 2760 may be different than the labeled probes in reservoir2730. As in process 2712, the labeled probes in reservoir 2760 maycouple to a subset of the individually addressable locations comprisingthe biological analyte e.g., based on sequence complementarity.Processes 2714, 2715 may then be repeated. In some cases, one or moreprocesses may be performed iteratively.

In some cases, the biological analyte is a nucleic acid molecule orclonal population of nucleic acid molecules, and the film 2710 is movedthrough a first reel to contact the film with a first reservoircomprising a plurality of adenine (e.g., fluorescently labeled adenine)molecules. The adenine molecules may then hybridize with a thyminemolecule within the biological analyte. The film may then be movedthrough the reel to contact the film with a wash reservoir to removeunhybridized probes. Detection of the hybridized molecules may occur.Since the sequence of the probe molecule is known, detection of one ormore signals may yield knowledge of the sequence of the biologicalanalyte. Subsequently, the film may then be brought into contact with areservoir comprising a labeled cytosine, a labeled guanine, or a labeledthymine, etc. Again, as each sequence of the probe is known, detectionof one or more signals may yield knowledge of the sequence of thebiological analyte. As will be appreciated, the specific nucleotideadded to each reservoir can vary; e.g., the first reservoir may comprisean adenine, cytosine, guanine, thymine, etc, and the next reservoir maycomprise an adenine, cytosine, guanine, thymine, etc.

As will be appreciated, any of the processes within the method describedherein may occur at any convenient step. For example, the flexiblesubstrate may first be brought into contact with a first reservoir,followed by a wash reservoir, followed by a second reservoir, prior todetection. In other examples, the flexible substrate may be brought intocontact with a plurality of reservoirs comprising probes prior todetection. In other examples, the flexible substrate may be brought intocontact with a detector prior to or following contacting the flexiblesubstrate with any number of reservoirs. Additionally, any number ofreels may be used. For example, it may be desirable to use a single reelfor an operation. In some cases, more than one reel may be used. Forexample, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more reels may beused.

In some cases, the detection method may comprise multi-channel imaging.

Immersion Optics

Disclosed herein, in certain embodiments, are systems for using opticalsensors, such as optical imaging objectives. The present disclosureprovides systems for modulation and management of temperature for one ormore systems or methods of the disclosure. In some embodiments of one ormore systems and methods described herein, an optical imaging objectiveis used during the detection method. In some cases, the optical imagingobjective is immersed in a fluid in contact with the substrate, and theoptical imaging objective is in optical communication with the detector.In some embodiments, the substrate performs optimally at a non-ambienttemperature (e.g., ˜50 degrees Celsius). In some cases, the opticalimaging objective may be close to ambient temperature. In such cases, asubstrate that is operating at a higher temperature (e.g., ˜50 degreeCelsius) may be in contact with the objective that operates at ambienttemperature (˜20 degrees Celsius), thereby generating a temperaturegradient between the substrate and the optical imaging objective. Insome cases, it may be desirable to control the temperature gradientlocation and the magnitude of the temperature gradient. Thus, providedherein are methods and systems for temperature modulation.

FIG. 16 illustrates schematically an exemplary temperature gradient thatmay arise between an optical imaging objective and a substrate. Theoptical imaging objective 1110 (e.g., as described with respect to FIG.15) may comprise a first element 2810, a second element 2830, a thirdelement 2840, and, in some cases, one or more spacers 2820. For example,the first element 2810 may comprise a front lens or a meniscus lens, thesecond element 2830 may comprise a first lens group such as a tripletlens group, and the third element 2840 may comprise a second lens groupsuch as a doublet lens group. Alternatively or in addition, the firstelement 2810 may comprise a planoconvex lens, the second element 2830may comprise a meniscus lens, and the third element 2840 may comprise anachromatic lens. The optical imaging objective 1110 may be at ambienttemperature. The substrate 310 may be a substrate described herein andmay comprise a biological analyte. In some cases, the substrate 310 isheated to a temperature that is greater than ambient temperature. Insome cases, the difference in temperature between the substrate 310 andthe optical imaging objective 1110 may generate a temperature gradient2850. The temperature gradient 2850 may result in heat transfer betweenthe substrate and the optical imaging objective 1110 as well as thesurrounding environment. In some cases, it may be desirable to modulateor regulate the temperature of the system or the substrate so that thesubstrate maintains a constant temperature.

FIG. 17A-FIG. 17E illustrate schematically example methods to regulatetemperature of the substrate. FIG. 17A illustrates an embodiment of sucha temperature regulation method of a system. The system may comprise asubstrate 310, which may be any substrate described herein, an opticalimaging objective 1110 as described herein, and an immersion fluid 1140.In some embodiments, it is desirable to maintain the substrate 310 at anelevated temperature (e.g., 50 degrees Celsius) while keeping othercomponents of the system (e.g., 2830, 2840, 2820) at ambienttemperature. In some cases, heat 2920 may be applied to the substrate310. The heat may transfer to other components of the system, such asthe immersion fluid 1140, and part of the optical imaging objective1110. In some cases, the first element 2810 of the optical imagingobjective 1110 may be robust to a large temperature gradient and may notbe critical to the optical path or detection method. In one non-limitingexample, the first element 2810 may be a substantially flat (e.g.,planar) surface. In such cases, the first element 2810 may be robust toa large temperature gradient and may not influence the optical path,detection, or magnification of the substrate or contents disposedthereof. In some cases, the heat 2920 applied to the substrate 310 maybe transferred conductively away from the optical imaging objective1110. For example, the heat 2920 applied to the substrate 310 maytransfer to the immersion fluid 1140, to the first element 2810, to theone or more spacers 2820, then toward the outer layer 2930 of theoptical imaging objective. The transferred heat may then travelconvectively away from the optical imaging objective 1110. In somecases, the heat may be transferred away from the optical imagingobjective and may travel from the substrate 310 to the immersion fluid1140, to the first element 2810. The heat may travel convectively to thesecond element 2830 and to the one or more spacers 2820 and may travelconvectively away from the optical imaging objective 1110. In someembodiments, the thermal resistance of one or more components of theoptical imaging objective 1110 may be modulated. For example, the outerlayer 2930 of the imaging optical imaging objective 1110 may beconfigured to optimally disperse heat (e.g., using brass or a lowresistivity material, designing thin layers, etc.).

In some embodiments, the method may comprise heating the immersionfluid. In some cases, the immersion fluid 1140 may be pre-heated andapplied to the substrate 310, so that the substrate maintains anelevated temperature (e.g., 50 degrees Celsius). The immersion fluid maybe continuously replenished. For example, the system may comprise afluid flow tube (e.g., 1130 in FIG. 15) that is configured to deliverimmersion fluid in an enclosed system. In such cases, the heat may betransferred away from the optical imaging objective via convection andconduction. In some cases, additional heat may be transferred away fromthe optical imaging objective using a cooling element 2910 a, such as afan, which may direct heat (e.g., convectively) away from the opticalimaging objective 1110 and reduce the temperature of the components ofthe optical imaging objective 1110.

FIG. 17B illustrates schematically another embodiment of a temperatureregulation method of a system. The system may comprise a substrate 310,as described herein, an optical imaging objective 1110, as describedherein, and an immersion fluid 1140. In some embodiments, the immersionfluid 1140 may be heated. In some embodiments, heat 2920 is added to thesubstrate 310. In some embodiments, the system comprises an insulatingspacer 2935, which may be configured to generate an insulated region2940, comprising the second element 2830 and the third element 2840,which is insulated from the elevated temperature region (e.g., the firstelement 2810, the immersion fluid 1140, and the substrate 310). In suchcases, the greatest temperature gradient may occur in the space betweenthe first element 2810 and the second element 2830. In some cases, theinsulating spacer 2935 may have a higher thermal resistance than glass.In some embodiments, a cooling element 2910 a may be used to furthercool the optical imaging objective 1110. In some embodiments, the firstelement 2810 may be configured to rapidly disperse heat (e.g., may bethin). In some embodiments, the insulating spacer 2935 may have a higherresistance than the first element 2810, which may reduce heat transferto the second 2830 and third 2840 elements. Alternatively or in additionto the insulating spacer, there may be a gap (e.g., air gap) disposedbetween the first element 2810 and the rest of the objective 1110. Insome embodiments, the first element 2810 may have optical propertiesthat are insensitive to temperature. In some embodiments, the firstelement 2810 may have zero or very low optical power, e.g., may be awindow or substantially flat (e.g., planar) element, thereby reducingthe sensitivity of the first element 2810 to temperature or thermallyinduced dimension fluctuations.

FIG. 17C illustrates schematically another embodiment of a temperatureregulation method of a system. The system may comprise a substrate 310,as described herein, an optical imaging objective 1110, as describedherein, an immersion fluid 1140, and a heating element 2910 b. In someembodiments, the optical imaging objective 1110 may be heated to adesired temperature (e.g., 50 degrees Celsius) or to a temperature tomatch the desired temperature of the substrate 310. In some cases,resistive heaters may be used for the optical imaging objective. Heatingof the optical imaging objective may result in heat transfer to thesubstrate 310. In some cases, heat 2920 may also be applied to thesubstrate 310. In some embodiments, the heating element 2910 b may beused to apply heat to the optical imaging objective, e.g., viaconvection.

FIG. 17D illustrates schematically another embodiment of a temperatureregulation method of a system. The system may comprise a substrate 310,as described herein, an optical imaging objective 1110, as describedherein, and an immersion fluid 1140. In some embodiments, the opticalimaging objective 1110 may be cooled. For example, cooled immersionfluid 1140 may be continuously circulated between the optical imagingobjective 1110 and the substrate 310. In some cases, the immersion fluid1140 may be recycled to minimize reagent use, as described elsewhereherein. In some embodiments, heat 2920 may be applied to the substrate310.

FIG. 17E illustrates schematically another embodiment of a temperatureregulation method of a system. The system may comprise a substrate 310,as described herein, an optical imaging objective 1110, as describedherein, and an immersion fluid 1140. In some embodiments, the opticalimaging objective 1110 may be cooled while the substrate 310 is heated.For example, cooled immersion fluid 1140 may be continuously circulatedbetween the optical imaging objective 1110 and the substrate 310. Insome cases, the flow rate of the immersion fluid 1140 may be controlledsuch that the temperature gradient 2850 exists primarily in theimmersion fluid 1140, and the immersion fluid 1140 close to thesubstrate is at an elevated temperature, but the immersion fluid 1140close to the optical imaging objective 1110 is cooled. In some cases,the immersion fluid 1140 may be recycled to minimize reagent use, asdescribed elsewhere herein.

As will be appreciated, any combination of mechanisms for temperatureregulation and/or modulation may be used. For example, the opticalimaging objective may comprise (i) an outer layer that may conduct heataway from the optical imaging objective and (ii) a flat or planar firstelement with zero or low optical power that is robust to temperature. Insome cases, the immersion fluid may be heated in addition oralternatively to using an optical imaging objective with a conductiveouter layer and/or flat first element. Similarly, a cooling element maybe implemented with any of the described methods and systems. Anysuitable combination of temperature modulation methods may be used inconjunction with the systems and methods described herein.

Also disclosed herein, in certain embodiments, are methods for fluid andbubble control in optical detection systems. In some embodiments, anoptical imaging objective is used during the detection method. In somecases, the optical imaging objective is immersed in a fluid in contactwith the substrate, and the optical imaging objective is in opticalcommunication with the detector. In some cases, the optical imagingobjective may comprise a camera or may be connected to a camera. In somecases, the camera or the optical imaging objective comprising the cameramay be in fluidic communication with the substrate. In some embodiments,the optical imaging objective or camera is located at a suitable workingdistance from the substrate. In some cases, the optical imagingobjective may be immersed in a fluid. In some embodiments, the opticalimaging objective or camera comprises an adapter that is configured tomaintain a fluid-filled cavity around the outlet of the optical imagingobjective or camera. In some cases, the adapter may allow for imaging ofthe substrate (or an uncovered surface thereof) at greater workingdistances. The adapter may be attached to or encase the optical imagingobjective or camera. In some cases, the adapter comprises a hydrophobicregion, such as the area that interfaces with the immersion fluid. Thehydrophobic region may allow for fluid to be directed towards or staynear the imaging region of the optical imaging objective. For example,the hydrophobic region may be configured to retain a volume of fluidbetween the optical imaging objective or camera and the imaged region ofthe substrate (or uncovered surface thereof). In some cases, the adaptercomprises a hydrophilic region, such as the area that interfaces withthe immersion fluid. The hydrophilic region may allow for fluid to bedirected towards or stay near the imaging region of the optical imagingobjective. For example, the hydrophilic region may be configured toretain a volume of fluid between the optical imaging objective or cameraand the imaged region of the substrate (or uncovered surface thereof).In some cases, the adapter comprises both a hydrophilic and ahydrophobic region, which may allow for fluid to be directed towards orstay near the imaging region of the optical imaging objective or camera.

FIG. 19 illustrates schematically an exemplary adapter that may beattached to or encase the optical imaging objective. The adapter 3100may allow for imaging of the substrate at greater working distances(e.g., greater than 500 microns). In some cases, the adapter simulates ashorter working distance by forming a fluid-filled cavity around theoptical imaging objective 1110. In some embodiments, the adapter 3100comprises one or more inlet ports 3110, which may dispense the immersionfluid. In some embodiments, the adapter 3100 also comprises one or moreother ports 3120 (e.g., outlet ports, additional inlet ports, etc.).Fluid may be directed to a cavity 3130 surrounding the optical imagingobjective 1110. In some cases, the fluid may be immersion fluid and maybe dispensed on the substrate 310. In some cases, the adapter 3100retains a volume of immersion fluid between the adapter and thesubstrate 310, e.g., via surface tension. Use of an adapter may allowfor greater working distances while maintaining immersion of the opticalimaging objective 1110 in the immersion fluid. In some cases, theadapter may comprise a hydrophobic region that allows for the immersionfluid to remain or be directed toward the imaging path of the opticalimaging objective 1110.

Suitable working distances between the optical imaging objective and thesubstrate may be any suitable distance for imaging the substrate. Insome cases, a working distance between 100 and 500 microns (μm) issuitable. For example, a suitable working distance may be 100, 150, 200,250, 300, 350, 400, 450, 500 microns. In some cases, a working distancemay be less than 100 microns. For example, a working distance may be 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, or 95 microns. In some cases, a working distance may be greaterthan 500 microns. For example, a suitable working distance may be 550,600, 650, 700, 750, 800, 850, 900, 950, or 1000 or more microns. In somecases, a suitable working distance may be more than 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500 or more microns. In some cases, the opticalimaging objective may be a long working distance objective. For example,the optical imaging objective may have a working distance of greaterthan 5, 6, 7, 8, 9, 10, 15, 20, 25 or more millimeters (mm).

In some cases, a working distance may be sufficiently small such that animmersion fluid may be retained (e.g., via surface tension) between theoptical imaging objective and the substrate. In some cases, a workingdistance may be greater, such that the immersion fluid does not touchthe optical imaging objective or the substrate. In some cases, anadapter may be added to the objective that can form a fluid-filledcavity around the objective, such that an immersion fluid may beretained (e.g., via surface tension) between the optical imagingobjective and/or adapter and the substrate.

In some embodiments, bubbles may form in the immersion fluid, which mayaffect the optical and/or detection performance of the system. Forexample, bubbles may form in the optical path of the optical imagingobjective, which may reduce the performance of imaging, focusing, andthe path of light (e.g., laser, LED, transmitted light, etc.). In somecases, it is desirable to prevent bubble formation and/or remove bubblesfrom the optical path. Thus, provided herein are methods and systems forpreventing formation of bubbles and for removal of bubbles from theoptical path.

FIG. 18 demonstrates schematically the formation of bubbles in animmersion fluid. An optical imaging objective 1110, as described herein,may be positioned over a substrate 310, such as a rotatable substrate, aplanar substrate, and/or any substrate described herein. Disposedbetween the optical imaging objective 1110 and the substrate 310 is animmersion fluid 1140, as described herein. In some cases, the immersionfluid may comprise bubbles 3010. The bubbles 3010 may occur along theoptical path of the optical imaging objective 1110, which may reduce theimaging performance of the detection method.

In some embodiments, the method may comprise substrate modification toprevent bubble formation. In some cases, the method comprises degassingthe immersion fluid before use in imaging. In some cases, the substratemodification may comprise immersion lithography. In some cases, ahydrophobic material, such as a resist, may be deposited onto thesurface of the substrate. Increasing the hydrophobicity of the substratemay increase the contact angle of a fluid on the surface of thesubstrate and reduce bubble formation.

In some cases, e.g., in immersion lithography, it may be desirable tominimize the exposure of the immersion fluid to the substrate. Thus, themethod may comprise methods to minimize the area and duration ofimmersion fluid contact with the substrate. In some embodiments, themethod comprises dispensing and recovery ports that dispense immersionfluid onto the substrate and remove the immersion fluid from thesubstrate, respectively. Recovery of the fluid may be obtained by avariety of means such as application of pressure or aspiration, gravityforces, centrifugal forces, capillary forces, electric forces, magneticforces, etc. In some cases, the dispensing and recovery parts may beused to minimize usage of reagents (e.g., immersion fluid). In suchcases, the immersion fluid may be recycled, as described elsewhereherein.

FIG. 21 illustrates schematically a method for dispensing and removingimmersion fluid onto a substrate. The substrate 310 may be any substratedescribed herein. The immersion fluid 1140 may comprise an imagingbuffer. In some cases, minimization of amount of immersion fluid may bedesired, or minimization of exposure of the substrate 310 to theimmersion fluid 1140 is desired. In some embodiments, the methodcomprises dispensing the immersion fluid 1140 through a dispensing port3210 and recovering the immersion fluid 1140 through a recovery port3220. In some cases, the dispensing port is located close to the opticalimaging objective 1110. In some cases, the recovery port is locatedoutside, i.e., radially outward, of the optical imaging objective 1110and the dispensing port 3210. In some cases, a plurality of dispensingand recovery parts may be used. As will be appreciated, any number ofdispensing and removal ports may be used. For example, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more dispensingports or removal ports may be used. In some embodiments, the number ofdispensing ports used may not be equal to the number of removal portsused. In some cases, more dispensing ports may be used than removalports. In other cases, more removal ports are used than dispensingports. In some embodiments, the dispensing and removal ports may be partof an adapter 3100 (see FIG. 19).

In some embodiments, the generation of bubbles may be minimized bycontrolling the flow rate of the immersion fluid. In some cases, e.g.,in immersion lithography, the flow rate of fluid dispensing may beoptimized. For example, the flow rate of fluid dispensing may be 1picoliter/min, 10 picoliters/min, 100 picoliters/min, 1 nanoliter/min,10 nanoliters/min, 100 nanoliters/min, 1 microliter/min, 10microliters/min, 100 microliters/min, 1 milliliter/min, 10milliliters/min, 100 milliliters/min, or up to 1 liter/min. The flowrate of fluid dispensing may be between any of these flow rates.Alternatively, the flow rate of fluid dispensing may be at most any ofthese flow rates. The flow rate may be sufficiently low such that bubblegeneration is minimized. In some embodiments, the flow rate may allowair or bubbles to rise above the objective and away from the opticalpath.

In some embodiments, the method may comprise dispensing a fluid on thesubstrate and then using the optical imaging objective to displacebubbles. FIG. 20A-FIG. 20B illustrate schematically a method to displacebubbles. In FIG. 20A, a substrate 310 may have dispensed thereto animmersion fluid 1140, as described herein. The immersion fluid 1140 maycomprise bubbles 3010. In FIG. 20B, the optical imaging objective 1110may be brought into contact with the immersion fluid 1140, thusdisplacing the bubbles 3010. In some embodiments, the optical imagingobjective 1110 may have attached thereto an adapter 3100 (not shown). Insome cases, the adapter 3100 may comprise a plurality of dispensing andrecovery ports. In such cases, the dispensing port or the recovery portmay be used to pull the fluid (e.g., via pressure differences, capillaryforces, etc.) into the adapter and thus away from the optical imagingobjective.

In some embodiments, the method may comprise using an adapter to preventbubble formation, or to trap or capture bubbles. As described herein,the adapter may be attached to the optical imaging objective. In somecases, the adapter may interface with the immersion fluid. In somecases, the adapter comprises dispensing ports that may dispense theimmersion fluid onto the substrate. In some embodiments, the surface ofthe adapter that interfaces with the immersion fluid may be flat. Insome cases, a thin layer of glass may be placed between the opticalimaging objective and the substrate to form a closed cavity to minimizebubble formation. In such an embodiment, the thin layer of glass may beplaced between the objective and the wafer to form a closed cavity. Theclosed cavity may be filled with an immersion liquid without bubbles. Onthe other end of the thin layer of glass, the fluid may be introducedbetween the thin layer of glass and the substrate.

In some embodiments, the adapter may be used to remove bubbles from theimmersion fluid. In some cases, the adapter comprises one or moredispensing and/or recovery ports. In some embodiments, the dispensingports may be used to rapidly flush immersion fluid onto the substrate,thereby breaking or disrupting larger bubbles into smaller bubbles,which may be cleared by a separate mechanism, or which may break. A highrapid flush may also push bubbles out of the adapter or away from theoptical imaging objective.

In some embodiments, the adapter may comprise ports that may be used toremove bubbles. For example, a suction (i.e., negative pressure) portmay be placed in the adapter that may attach to the optical imagingobjective. In some embodiments, the suction port may be used to removebubbles in the vicinity. In other cases, the adapter may comprise adispensing port that rapidly dispenses fluid onto the substrate to movebubbles toward another area of the substrate. The adapter in some casesmay also comprise a suction port to aspirate the bubbles. As will beappreciated, any combination of the features of the adapter (e.g.,dispensing port, recovery port, suction ports) may be used.

In some embodiments, the adapter may be flat relative to the plane inwhich the adapter interfaces with the immersion fluid, for example asshown in FIG. 22A. In some embodiments, the adapter may be convex alongthe plane or area that interfaces with the immersion fluid, for exampleas shown in FIG. 22B. In some cases, the bottom surface of the adaptermay interface with the immersion fluid and may be partly angled, e.g.,in a cone shape. The angled shape may reduce the area of contact betweenthe immersion fluid and the adapter. In some cases, the angled shape mayguide or direct fluid to the optical path. In some cases, the opticalimaging objective may be the closest part to the substrate and/orimmersion fluid. In some embodiments, the adapter may be asymmetrical inshape to reduce the area of the adapter in contact with the immersionfluid.

In some embodiments wherein the adapter is angled, the angle between theadapter and the immersion fluid may be any suitable angle. The angle maybe, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60 degrees. In some cases, the angle may beat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60 degrees. In some cases, the angle may be at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60 degrees. In some cases, the angle may be a non-integerangle.

In some embodiments, the adapter may comprise a trap that may captureand remove bubbles from the optical path. For example, the adapter maycomprise a cavity that may direct bubbles into an internal region of theadapter. Alternatively, the cavity may be connected to an outlet portthat allows for disruption of the bubble or removal of the bubble.

FIG. 22A-FIG. 22B illustrate schematically a method for trappingbubbles. FIG. 22A illustrates an exemplary adapter 3100, as describedherein, which encases an optical imaging objective 1110, as describedherein. The adapter 3100 may be flat or may be angled (see, FIG. 22B).The adapter 3100 may interface with an immersion fluid 1140, which maycomprise bubbles 3010. The adapter 3100 may comprise a cavity that cancapture entrained bubbles 3010. In some cases, the bubbles may disrupt,break, or pop in the cavity. In other cases, the cavity may be connectedto a port (not shown). In FIG. 22B, the adapter may have an angledbottom, which may reduce the area of contact between the immersion fluid1140 and the adapter 3100. The angle, θ, may be any suitable or usefulangle.

In some cases, one or more components of the system may be moved (e.g.,translated) to remove bubbles. In one non-limiting example, the opticalimaging objective may be moved vertically away from the substrate andthen repositioned to an imaging position, thereby allowing entrainedbubbles to displace and/or break. In some cases, the substrate may bemoved relative to the objective, thereby allowing entrained bubbles todisplace and/or break. In another non-limiting example, the substratemay be moved in the plane, e.g., in a circular motion or linear motion(e.g., as shown in FIG. 23A-FIG. 23J). In some cases, motion of thesubstrate may generate a shear force and velocity field that causesbubbles to displace and/or break. In some cases, a combination of motionplanes may be employed. For example, either the optical imagingobjective or the substrate, or both, may be moved both in a vertical andplanar direction. At any step in the motion, an immersion fluid may bedispensed onto the substrate.

In some embodiments, the immersion fluid may be recollected and recycled(or recirculated). In some cases, the immersion fluid may be treatedprior to recycling or recirculation. Treatment may comprise removingdebris, removing analytes (e.g., nucleotides, proteins, lipids,carbohydrates, etc.), removing beads, or any other contaminants.Treatment may comprise degassing, de-bubbling, or removing entrainedair. As will be appreciated any treatment may comprise any combinationof these processes in any convenient order.

Optical Layouts

The present disclosure provides optical systems that are designed toimplement the methods of the disclosure. FIG. 41 shows an exemplaryoptical system that may be used to scan a substrate as disclosed herein,for example a rotating substrate. The optical system may comprise one ormore distinct optical paths. The one or more optical paths may comprisemirrored optical layouts. In some embodiments, the optical system maycomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 distinctoptical paths. For example, the optical system may comprise two distinctoptical paths, as shown in FIG. 41.

An optical path may comprise an excitation path and an emission path.The excitation path and the emission path may each comprise a pluralityof optical elements in optical communication with the substrate. In someembodiments, the excitation path comprises one or more of an excitationlight source, a beam expander element, a line shaper element, adichroic, and an objective. In some embodiment, the emission path maycomprise one or more of an objective, a dichroic, a tube lens, and adetector. The objective in the excitation path may be the same as theobjective in the emission path. The objective may be an immersionobjective, or the objective may be an air objective. In someembodiments, the objective is immersed in water, buffer, aqueoussolution, oil, organic solvent, index matching fluid, or other immersionfluid. The objective may be a 10×, 20×, 50×, or 100× objective.

The dichroic in the excitation path may be the same as the dichroic inthe emission path. The dichroic may be a short pass dichroic, or thedichroic may be a long pass dichroic. In some embodiments, the dichroicpasses the excitation light and reflects the emission light. In otherembodiments, the dichroic reflects the excitation light and passes theemission light. The dichroic may have a cutoff wavelength of about 250nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1000nm, about 1050 nm, or about 1100 nm. The dichroic may have a cutoffwavelength of from 250 nm to 300 nm, from 300 nm to 350 nm, from 350 nmto 400 nm, from 400 nm to 450 nm, from 450 nm to 500 nm, from 500 nm to550 nm, from 550 nm to 600 nm, from 600 nm to 650 nm, from 650 nm to 700nm, from 700 nm to 750 nm, from 750 nm to 800 nm, from 800 nm to 850 nm,from 850 nm to 900 nm, from 900 nm to 950 nm, from 950 nm to 1000 nm,from 1000 nm to 1050 nm, from 1050 nm to 1100 nm, from 250 nm to 400 nm,from 350 nm to 500 nm, from 450 nm to 600 nm, from 550 nm to 700 nm,from 650 nm to 800 nm, from 750 nm to 900 nm, from 850 nm to 1000 nm, orfrom 950 nm to 1100 nm.

The excitation light source may be configured to emit light, for examplecoherent light. The excitation light source may comprise one or morelight emitting diodes (LEDs). The excitation light sources may compriseone or more lasers. The excitation light sources may comprise one ormore single-mode laser sources. The excitation light sources maycomprise one or more multi-mode laser sources. The excitation lightsources may comprise one or more laser diodes. A laser may be acontinuous wave laser or a pulsed laser. A beam of light emitted by alaser may be a Gaussian or approximately Gaussian beam, which beam maybe manipulated using one or more optical elements (e.g., mirrors,lenses, prisms, waveplates, etc.). For example, a beam may becollimated. In some cases, a beam may be manipulated to provide a laserline (e.g., using one or more Powell lenses or cylindrical lenses). Theexcitation light source may be coupled to an optical fiber.

The line shaper may be configured to expand the excitation light sourcealong one axis, for example as shown in FIG. 11A and FIG. 11B. The lineshaper may comprise one or more lenses. In some embodiments, the lineshaper comprises one or more cylindrical lenses. The one or morecylindrical lenses may be convex cylindrical lenses, concave cylindricallenses, or any combination thereof. In some embodiments, the line shaperis positioned in a rotating mount, for example a motorized rotatingmount. The rotational mount may be configured to rotate the expandedexcitation light source about a central axis without substantialdeviation of the central point of the excitation light source. In someembodiments, the line shaper element may be configured to rotate aboutthe central axis in response to, concurrent with, or in anticipation ofa translation of the substrate with respect to the optical system. Forexample, the line shaper element may rotate about the central axis suchthat the axis of the expanded excitation light maintains a definedorientation with respect to the rotational axis of the substrate upontranslation of the substrate with respect to the optical axis in adirection that is not directly toward or away from the rotational axis.

The beam expander may comprise one or more lenses. For example, the beamexpander may comprise two lenses. The lenses may have different focallengths. In some embodiments, the lens closer to the excitation lightsource may have a shorter focal length that the lens farther from theexcitation light source. The beam expander may be configured to expandthe excitation light source about 2×, about 3×, about 4×, about 5×,about 10×, about 15×, or about 20×. The beam expander may be configuredto collimate the excitation light source. The beam expander may beconfigured to focus the excitation light source.

The tube lens may comprise one or more lenses. For example, the tubelens may comprise two lenses. The lenses may have different focallengths, or the two lenses may have different focal lengths. The tubelens may be configured to expand the excitation light source about 2×,about 3×, about 4×, about 5×, about 10×, about 15×, or about 20×. Thetube lens may be configured to collimate the emission light. The tubelens may be configured to focus the emission light.

The detectors may comprise any combination of cameras (e.g., CCD, CMOS,or line-scan), photodiodes (e.g., avalanche photo diodes),photoresistors, phototransistors, or any other optical detector known inthe art. In some embodiments, the detectors may comprise one or morecameras. For example, the cameras may comprise line-scan cameras, suchas TDI line-scan cameras. In some embodiments, the TDI line-scan cameramay comprise two or more vertically arranged rows of pixels, as shownwith respect to FIG. 8A-FIG. 8D. The detector may be configured torotated with respect to the substrate to correct for tangential velocityblur, as described herein. In some embodiments, the detector may beconfigured to rotate in response to, concurrent with, or in anticipationof a translation of the substrate with respect to the optical system.For example, the detector may rotate such that the axis of the imagingfield maintains a defined orientation with respect to the rotationalaxis of the substrate upon translation of the substrate with respect tothe optical axis in a direction that is not directly toward or away fromthe rotational axis. The detector may be configured to rotateconcurrently with a rotation of the line shaper element, such that theimaging field maintains a defined orientation with respect to the axisof the expanded excitation light. The detector may be configured torotate independently of the line shaper element.

The optical path may comprise additional optical components not shown inFIG. 41. For example, an optical path may comprise additional splitting,reflecting, focusing, magnifying, filtering, shaping, rotating,polarizing, or other optical elements.

One or more optical elements in the optical path may be positioned in amount. A mount may be a rotational mount. A mount may be a kinematicmount. A mount may be a translational mount. A mount may be a stationarymount. In some embodiments, a mount may have one or more degrees offreedom. For example, a mount may have one or more of one-dimensionaltranslation, two-dimensional translation, three-dimensional translation,one dimensional rotation, two-dimensional rotation, or three-dimensionalrotation.

The optical systems of this disclosure may further comprise one or moreautofocus systems (not shown in FIG. 41). In some embodiments, eachoptical path in the optical system comprises an autofocus system. Theautofocus system may comprise an autofocus illumination sourceconfigured to direct autofocus light through the objective toward thesurface. In some embodiments, the autofocus illumination source maycomprise an infrared (IR) laser, for example, a speckle-free IR laser.The autofocus light may pass through one or more of the optical elementsin the optical path. In some embodiments, the optical path comprises oneor more optical elements to differentially reflect or combine one ormore of the excitation light, the emission light, or the autofocuslight. The one or more optical elements may comprise one or moredichroics. The autofocus light may reflect, refract, or scatter off thesurface toward an autofocus detector. The autofocus detector may be aposition-sensitive detector. The autofocus light may coincide with theautofocus detector at a discrete position when the surface is in focuson an emission detector (e.g., the camera illustrated in FIG. 41). Theautofocus illumination source and the autofocus detector may beconfigured such that a change in a position of the surface relative tothe objective results in a change in position of the autofocusillumination on the autofocus detector. For example, a change in adistance between the surface and the objective or a tilt of the surfacerelative to the objective may cause a displacement of the autofocusillumination position on the autofocus detector. The autofocus systemmay send a signal to a focusing system in response to the change inposition of the autofocus illumination on the autofocus detector. Thefocusing system may adjust the position of the surface relative to theobjective such that the position of the autofocus illumination on theautofocus detector returns to the discrete position when the surface isin focus on the emission detector.

The optical systems of this disclosure may be aligned such that theexcitation light and the emission light pass substantially through thecenter of the optical elements. In some embodiments, the excitationlight may be aligned with respect to the line shaper element such thatthe position of the excitation light after passing through the lineshaper does not change substantially upon rotation of the line shaper.The line shaper may be rotated during alignment and the position of theexcitation light source, the line shaper, or both may be adjusted tominimize motion of the position of the excitation light after passingthrough the line shaper upon rotation of the line shaper. In someembodiments, a position of the detector is aligned with respect to arotating mount. For example, the detector is centered within therotational mount by illuminating the center of the detector, rotatingthe rotational mount, and adjusting the position of the detector withinthe mount so that the position of the illumination does not move uponrotation of the rotational mount. In some embodiments, the position ofthe excitation light is aligned at two or more points thereby definingboth a position and an angle. In some embodiments, the position of theemission light is aligned at two or more points thereby defining both aposition and an angle.

The one or more imaging heads of this disclosure may be aligned withrespect to the substrate. In some embodiments, the positions of the oneor more imaging heads are adjusted in zero, one, two, or threetranslational dimensions (e.g., x, y, and z) and zero, one, two, orthree rotational dimensions (e.g., α, β, and γ). In some embodiments,the position of one or more optical elements may be adjusted in anycombination of translational or rotational dimensions. The opticalsystems of this disclosure may be coarsely aligned at low excitationpower. The alignment of the optical systems of this disclosure may beprecisely aligned at higher excitation powers. In some embodiments, thealignment of the optical systems may change upon increase of theexcitation power. In some embodiments, the optical system may be alignedduring one or more of rotation of the substrate, translation of thesubstrate, or translation of one or more imaging heads. The opticalsystems of this disclosure may be aligned using any alignment methodknown in the art.

Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 1 shows acomputer system 101 that is programmed or otherwise configured tosequence a nucleic acid sample. The computer system 101 can regulatevarious aspects of methods and systems of the present disclosure.

The computer system 101 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 105, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 101 also includes memory or memorylocation 110 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 115 (e.g., hard disk), communicationinterface 120 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 125, such as cache, other memory,data storage and/or electronic display adapters. The memory 110, storageunit 115, interface 120 and peripheral devices 125 are in communicationwith the CPU 105 through a communication bus (solid lines), such as amotherboard. The storage unit 115 can be a data storage unit (or datarepository) for storing data. The computer system 101 can be operativelycoupled to a computer network (“network”) 130 with the aid of thecommunication interface 120. The network 130 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 130 in some cases is atelecommunication and/or data network. The network 130 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. The network 130, in some cases with the aid of thecomputer system 101, can implement a peer-to-peer network, which mayenable devices coupled to the computer system 101 to behave as a clientor a server.

The CPU 105 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 110. The instructionscan be directed to the CPU 105, which can subsequently program orotherwise configure the CPU 105 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 105 can includefetch, decode, execute, and writeback.

The CPU 105 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 101 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 115 can store files, such as drivers, libraries andsaved programs. The storage unit 115 can store user data, e.g., userpreferences and user programs. The computer system 101 in some cases caninclude one or more additional data storage units that are external tothe computer system 101, such as located on a remote server that is incommunication with the computer system 101 through an intranet or theInternet.

The computer system 101 can communicate with one or more remote computersystems through the network 130. For instance, the computer system 101can communicate with a remote computer system of a user. Examples ofremote computer systems include personal computers (e.g., portable PC),slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab),telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device,Blackberry®), or personal digital assistants. The user can access thecomputer system 101 via the network 130.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 101, such as, for example, on the memory110 or electronic storage unit 115. The machine executable ormachine-readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 105. In some cases, thecode can be retrieved from the storage unit 115 and stored on the memory110 for ready access by the processor 105. In some situations, theelectronic storage unit 115 can be precluded, and machine-executableinstructions are stored on memory 110.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code or can be compiled duringruntime. The code can be supplied in a programming language that can beselected to enable the code to execute in a pre-compiled or as-compiledfashion.

Aspects of the systems and methods provided herein, such as the computersystem 101, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 101 can include or be in communication with anelectronic display 135 that comprises a user interface (UI) 140 forproviding, for example, nucleic acid sequencing information to a user.Examples of UI's include, without limitation, a graphical user interface(GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 105.

EXAMPLES Example 1. Imaging of Sequencing of a Nucleic Acid Molecule

FIG. 42 shows an example of an image generated by imaging a substratewith an analyte immobilized thereto. A substrate 310 comprising asubstantially planar array has immobilized thereto the biologicalanalyte, e.g., nucleic acid molecules. The substantially planar arraycomprises a plurality of individually addressable locations 320, and aplurality of the individually addressable locations comprises abiological analyte, e.g., one or more nucleic acid molecules. Theindividually addressable locations 320 may be randomly arranged orarranged in an ordered pattern. The biological analyte may be attachedto a bead, which is immobilized to the array. A single bead may comprisea plurality of analytes, such as at least 10, 20, 30, 40, 50, 100, 150or more analytes. A bead may be associated with an individuallyaddressable location. A plurality of fluorescent probes (e.g., aplurality of fluorescently-labeled, A, T, C, or G) is dispensed onto thesubstrate 310. In some embodiments, the substrate is configured torotate with respect to a central axis; a fluid flow unit comprising afluid channel configured to dispense a solution comprising a pluralityof probes to the array, wherein during rotation of the substrate, thesolution is directed centrifugally along a direction away from thecentral axis and brought in contact with the biological analyte. Inother embodiments, the substrate is not rotated. The substrate 310 isthen subjected to conditions sufficient to conduct a reaction between atleast one probe of the plurality of probes and the biological analyte,to couple the at least one probe to the biological analyte. Theuncoupled probes are washed away. The coupling of the at least one probeto the biological analyte is detected using photometry, which comprisesimaging at least a part of the substrate 310 (e.g., via scanning orfixed field imaging) and measuring the signal of each individuallyaddressable location 320. Nucleic acid molecules comprising a nucleotidecomplementary to the fluorescent probes are fluorescent in anindividually addressable location 320. The operations may then beiterated, and signals from an image are collated with signals from priorimages of the same substrate to generate traces of signals in time foreach biological analyte in each individually addressable location 320.The sequence of the plurality of fluorescent probes is known for eachiteration of the operations, generating a known sequence for the analytein each of the individually addressable locations 320.

Example 2. Diagnostic Procedure for Nucleic Acid Incorporation

Diagnostic procedures are run to determine whether a probe has coupledwith a biological analyte (e.g., nucleic acid molecule). FIG. 43 showsexample data of such a diagnostic procedure, running approximately 29giga base pairs (Gbp) from about 183 million beads. A substrate, similarto that depicted in 310 for example in FIG. 15-FIG. 23, comprises anarray configured to immobilize the biological analyte. The biologicalanalyte may be attached to a bead, which is immobilized to the array. Asingle bead may comprise a plurality of analytes, such as at least 10,20, 30, 40, 50, 100, 150 or more analytes. The biological analyte insome cases is genomic DNA from E. Coli bacteria. In some cases, humanDNA may be used as the biological analyte. In some cases, the biologicalanalyte is a shotgun library of DNA from a clonal population. In somecases, the substrate is configured to rotate with respect to a centralaxis. In other embodiments, the substrate is not configured to rotateand may be stationary. In other embodiments, the substrate is notconfigured to rotate and may be movable laterally or longitudinally, asdescribed elsewhere herein. In some cases, a fluid flow unit comprisinga fluid channel is used to dispense a solution comprising a plurality ofprobes (e.g., fluorescently labeled nucleotides) to the array, whereinduring rotation of the substrate, the solution is directed centrifugallyalong a direction away from the central axis and brought in contact withthe biological analyte under conditions sufficient to couple at leastone probe (e.g., nucleotide) of the plurality of probes to thebiological analyte. In other cases, the probes may be dispensed on thesubstrate via nebulization, a spray, a pressurized gas (e.g., blown gas)system, etc., as described elsewhere herein. The substrate 310 is thensubjected to conditions sufficient to conduct a reaction between atleast one probe of the plurality of probes and the biological analyte,to couple the at least one probe to the biological analyte. Theuncoupled probes are washed away. The coupling of the at least one probeto the biological analyte is detected using photometry, which comprisesimaging at least a part of the substrate. Nucleic acid moleculescomprising a nucleotide complementary to the fluorescent probes arefluorescent in an individually addressable location. One or more of theprocesses may be repeated or iterated in a cycle.

From the images, the signal 2320 of each individually addressablelocation or a plurality of individually addressable locations ismeasured. The mean signal 2330 of multiple individually addressablelocations can also be obtained for each cycle. Since the probe appliedto the substrate is known each cycle, the mean signal 2330 can beplotted as a function of the known nucleotide sequence 2310.Additionally, the standard deviation of the signal 2340 can also beplotted for each cycle. The plot 2300 may then yield information on thenucleic acid sequence of the biological analyte. One or more of theseoperations may be performed in real time.

Example 3. Scanning Image Pattern of a Biological Analyte

FIG. 44 shows example data of a diagnostic procedure that informsquality control metrics of scanning imaging. A substrate, similar tothat depicted in 310, may be subjected to rotation. The substrate insome cases is rotatable with respect to a central axis. In otherembodiments, the substrate may not be rotatable or may not be rotated.The substrate comprises the biological analyte, such as human and E.Coli shotgun libraries. In one example, the substrate comprises ashotgun library and ˜15% synthetic monotemplates that are spiked intothe sample. In such an example, the shotgun library and syntheticmonotemplates may be labeled (e.g., fluorescently). In other examples,the shotgun library and synthetic monotemplates are associated with abead, which may associate with the substrate (e.g., via a linker). Insome cases, the beads may associate with the substrate in a pattern. Insome cases, a subset of beads on the substrate may be detected in apattern, such as a spiral pattern (e.g., according to a scan path). Thelibrary and synthetic monotemplates may be detected directly using anoptical measurement. In other examples, a plurality of probes is addedto the substrate and the substrate is subjected to conditions sufficientto conduct a reaction between at least one probe of the plurality ofprobes and the biological analyte, to couple the at least one probe tothe biological analyte. One or more signals are detected from the atleast one probe coupled to the biological analyte.

Diagnostic metrics may be computed of imaged segments. FIG. 44A-FIG. 44Fshow plots depicting image or process metrics at different individuallyaddressable locations (e.g., varying R and θ on a circular substrate).Each scan field of view is depicted as a small circle on each plot(Panels A-F). The images may then be analyzed for the number of readsper image (Panel A), percentage of reads passing filter (Panel B), meanfirst incorporation signal of a nucleotide (Panel C), droop (signal lossper cycle, Panel D), lag phasing, which may be indicative of falsenegatives, e.g., the fraction of the clonal population that fails toadvance per cycle (Panel E), and lead phasing, which may be indicativeof false positives, e.g., the fraction of the clonal population thatincorrectly advances per cycle (Panel F). Uniform signal level andlead/lag phasing across R and θ indicate consistent fluidic andbiochemical reactions over the course of many incorporation cycles inthis instance and predict high quality sequence reads.

Example 4. Linearity and Accuracy of Homopolymers

In sequencing by synthesis chemistries based on single nucleotide flowsit is necessary to determine the length of hompolymers as they aresynthesized in order to determine the sequence. A homopolymer can be ofvarying lengths and comprise a sequence of identical nucleotides (e.g.,one nucleotide, two nucleotides, three nucleotides, four nucleotides,five nucleotides, six nucleotides, seven nucleotides, eight nucleotides,nine nucleotides, and ten nucleotides, wherein the nucleotides are allthe same, i.e., all A, all T, all C, all G, etc.). FIG. 45A showsexemplary data of flow-based sequencing by synthesis. Many homopolymersof different lengths were coupled to the substrate. A complementaryprobe was added to the substrate, and the substrate was washed andimaged, and the process was repeated. Signal was measured from each beadposition. As can be visualized in the plot, the signals from the imagesare quite linear with the homopolymer length, up to the maximum of 9nucleotides tested here. Thus, the signal from the obtained images(e.g., of an individually addressable location) can be used to determinethe homopolymer length up to 5 bases with sufficiently high accuracy andlow noise (>99% accuracy).

Example 5. Sequencing of Nucleic Acid Molecules and Signal Processing

A substrate comprising a substantially planar array has immobilizedthereto the biological analyte, e.g. nucleic acid molecules from E.coli. Sequencing by synthesis was performed using a flow-basedchemistry. Imaging was performed, as described elsewhere herein. FIG.45B shows the signal distributions for a set of several hundredcolonies, each a replicate of a single synthetic monotemplate. Thex-axis is labeled with the length of the sequencing after each cycle(e.g., each chemistry flow step). In FIG. 45C, the same data have beenprocessed with a parametric model. The parametric model accounts fordifferent template counts (amplitude) and background level for eachcolony. The signal is deconvolved with a model of lead and lag phasingand also accounts for global signal loss per cycle. In the exampledepicted here, the nominal phasing was 0.54% lag, 0.41% lead, and asignal loss of 0.45%. The residual systematic variation may beattributable to signal variation with sequence context can be furthercorrected using other algorithms (not shown).

Example 6. Sequencing of Shotgun Library from E. coli

A substrate comprising a substantially planar array has immobilizedthereto the biological analyte, e.g. nucleic acid molecules from E.coli. Sequencing by synthesis was performed using a flow-basedchemistry. Imaging was performed, as described elsewhere herein. Imageswere then processed. FIG. 46A shows individual aligned reads for asegment of the E. coli reference genome. FIG. 46B shows a plot derivedfrom the image processing of the aligned read depth for each position inthe E. coli genome for a set of shotgun reads. The x-axis shows thecoverage level at each E. coli reference key position and the y-axisshows the frequency.

Example 7. Calculation of Reel-to-Reel Dimensions

A flexible substrate comprising a biological analyte may be designed ina manner such that the throughput of processing nucleic acid moleculesis improved. In one example, biological analytes are nano-imprinted on aflexible substrate, such as a film, that is pulled through a first reelto contact the flexible substrate with a reservoir comprising a solutioncomprising a plurality of probes. The dimensions of the film may bemodulated to be compatible with the detector (e.g., an optical sensor).In some cases, the length of the film may be rolled around a reel. Thefilm may be ˜85 meters long and 7 millimeters (mm) wide, yielding anarea of ˜6000 square centimeters (cm²). Compared to a planar, circularsubstrate that has a diameter of 5.9 centimeter (cm), the usable area ofthe film may be over 60 times greater than the usable area of theplanar, circular substrate. Given an optical sensor rate of 10centimeters per second (cm/s), the entire film may be imaged within ˜14minutes. Alternatively, the dimensions (e.g., length and width) of thefilm may be modulated to improve the detection rate, the imprintingrate, the contact area, etc.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for analyzing a biological material,comprising: (a) activating a device comprising (i) a substratecomprising a surface having said biological material, wherein saidsurface is at a first temperature that is greater than an ambienttemperature, (ii) an optical imaging objective in optical communicationwith said surface, wherein said optical imaging objective comprises atemperature gradient between said first temperature and said ambienttemperature, wherein said optical imaging objective comprises (1) afirst optical element that is at least partially immersed in animmersion fluid in contact with said surface, and (2) a second opticalelement in optical communication with said surface through at least saidfirst optical element, and wherein said second optical element ismaintained at or below a second temperature different from said firsttemperature, wherein said optical imaging objective comprises a spacerbetween said first optical element and said second optical element,which spacer comprises a conductive material; and (b) using said opticalimaging objective to collect a signal from said surface having saidbiological material.
 2. The method of claim 1, wherein said firstoptical element is a window in optical communication with said surfaceand said second optical element.
 3. The method of claim 2, wherein saidwindow is substantially flat.
 4. The method of claim 1, wherein saidbiological material is a nucleic acid molecule.
 5. The method of claim2, wherein said window is substantially insensitive to thermally induceddimension fluctuations.
 6. The method of claim 1, wherein said secondoptical element is a lens.
 7. The method of claim 1, wherein saidoptical imaging objective comprises a housing enclosing said firstoptical element and said second optical element.
 8. The method of claim7, wherein said optical imaging objective comprises a heat flux paththat conducts heat between said substrate and an environment surroundingsaid substrate through said optical imaging objective.
 9. The method ofclaim 8, wherein said heat flux path does not pass through said secondoptical element.
 10. The method of claim 1, wherein said firsttemperature is at least 50 degrees Celsius.
 11. The method of claim 1,wherein said second temperature is said ambient temperature.
 12. Themethod of claim 1, wherein said second temperature is at most about 25degrees Celsius.
 13. The method of claim 1, wherein at least a portionof said first optical element is within 5 degrees Celsius of said firsttemperature.
 14. The method of claim 1, wherein said device furthercomprises a fluid flow unit that exchanges said immersion fluid incontact with said surface and said first optical element.
 15. The methodof claim 14, wherein said fluid flow unit maintains said immersion fluidat a third temperature, wherein said third temperature is within 5degrees Celsius of said first temperature.
 16. The method of claim 1,further comprising provididing a fluid flow unit that dispenses saidimmersion fluid to said surface, wherein said fluid flow unit dispensessaid immersion fluid at a rate of less than about 1 milliliter persecond.
 17. The method of claim 1, further comprising providing a fluidflow unit that dispenses said immersion fluid to said surface, whereinsaid fluid flow unit dispenses said immersion fluid to said surfacewhile said optical imaging objective is not contact with said immersionfluid.
 18. The method of claim 1, wherein said device further comprises(i) a container that at least partially encloses said optical imagingobjective, and (ii) a pressure unit that draws in a volume of saidimmersion fluid from outside said container into said container whilesaid optical imaging objective is in contact with said immersion fluid.19. The method of claim 1, further comprising providing a container thatat least partially encloses said optical imaging objective, wherein awall of said container contacts said immersion fluid, wherein said wallis angled with respect to a wall of said first optical element.
 20. Themethod of claim 1, further comprising providing a casing that at leastpartially encloses said first optical element, wherein said casingcomprises a cavity adjacent to said first optical element, wherein saidcavity contacts said immersion fluid and directs one or more bubbles insaid immersion fluid away from said first optical element.
 21. Themethod of claim 20, wherein said cavity at least partially surroundssaid first optical element.
 22. The method of claim 1, furthercomprising providing a movement unit operatively coupled to saidsubstrate or said optical imaging objective, wherein said movement unitmoves said substrate and said optical imaging objective relative to oneanother.
 23. The method of claim 22, wherein movement by said movementunit comprises a vertical component that is substantially perpendicularto a plane of said surface and a horizontal component that issubstantially parallel to a plane of said surface.
 24. A method foranalyzing a biological material, comprising: (a) activating a devicecomprising (i) a substrate comprising a surface having said biologicalmaterial, wherein said surface is at a first temperature that is greaterthan an ambient temperature, (ii) an optical imaging objective inoptical communication with said surface, wherein said optical imagingobjective comprises a temperature gradient between said firsttemperature and said ambient temperature, wherein said optical imagingobjective comprises (1) a first optical element that is at leastpartially immersed in an immersion fluid in contact with said surface,and (2) a second optical element in optical communication with saidsurface through at least said first optical element, and wherein saidsecond optical element is maintained at or below a second temperaturedifferent from said first temperature, and wherein said optical imagingobjective comprises an insulating spacer disposed between said firstoptical element and said second optical element, wherein said insulatingspacer reduces heat transfer from said first optical element and saidsecond optical element; and (b) using said optical imaging objective tocollect a signal from said surface having said biological material. 25.The method of claim 24, wherein said insulating spacer has a thermalresistance higher than a thermal resistance of said first opticalelement.
 26. A method for analyzing a biological material, comprising:(a) activating a device comprising (i) a substrate comprising a surfacehaving said biological material, wherein said surface is at a firsttemperature that is greater than an ambient temperature, (ii) an opticalimaging objective in optical communication with said surface, whereinsaid optical imaging objective comprises a temperature gradient betweensaid first temperature and said ambient temperature, wherein saidoptical imaging objective comprises (1) a first optical element that isat least partially immersed in an immersion fluid in contact with saidsurface, and (2) a second optical element in optical communication withsaid surface through at least said first optical element, and whereinsaid second optical element is maintained at or below a secondtemperature different from said first temperature, and wherein saidoptical imaging objective comprises a cooling element that cools saidsecond optical element; and (b) using said optical imaging objective tocollect a signal from said surface having said biological material. 27.A system for analyzing a biological material, comprising: a platformconfigured to support a substrate comprising a surface having saidbiological material, wherein said surface is configured to be at a firsttemperature that is greater than an ambient temperature when saidsubstrate is supported by said platform; an optical imaging objectiveconfigured to be in optical communication with said surface when saidsubstrate is supported by said platform, wherein said optical imagingobjective is configured to comprise a temperature gradient between saidfirst temperature and said ambient temperature, wherein said opticalimaging objective comprises (1) a first optical element that isconfigured to be at least partially immersed in an immersion fluid incontact with said surface, and (2) a second optical element in opticalcommunication with said surface through at least said first opticalelement, and wherein said second optical element is configured to bemaintained at or below a second temperature different from said firsttemperature, wherein said optical imaging objective comprises a spacerbetween said first optical element and said second optical element,which spacer comprises a conductive material; and one or more computerprocessors that are individually or collectively programmed to directcollection of a signal from said surface having said biological materialusing at least said optical imaging objective.
 28. The system of claim27, wherein said one or more computer processors are individually orcollectively programmed to maintain said surface at said firsttemperature.
 29. The system of claim 27, wherein said one or morecomputer processors are individually or collectively programmed todirect dispensing of said immersion fluid such that said immersion fluidcomes in contact with said surface.